#3E-Green Movement

[casbooks]

Books of 2023

Book 22 of 2023

Title: The Ravens: The Men Who Flew in America's Secret War in Laos Authors: Christopher Robbins ISBN: 9780517566121 Tags: A-1 Skyraiders, AC-47 Spooky, Ambassador Leonard Unger, Antonov AN-2 Colt, Aviation, B-52 Stratofortress, C-130 Hercules, C-46 Commando, C-47 Skytrain, CH-34 Choctaw, CIA Allen Dulles, CIA Hugh Tovar, COD Democratic Republic of the Congo - Congo-Kinshasa, COD Kinshasa (Leopoldville), COD Lubumbashi (Elisabethville), COD MNC Congolese National Movement, COD Mobutu Sese Seko, COD Patrice Lumumba, COD Simba Rebellion (1963-1965), CSAR, EC-47 Electric Goon, F-105 Thunderchief, F-4 Phantom II, FAC, Fast-FAC, FRA ADT Colonel Roger Trinquier, FRA ADT French Ground Army (Armée de terre), FRA ADT General Henri Navarre, FRA ADT General Raoul Salan, FRA ADT Marshall Jean de Lattre de Tassigny, FRA France, FRA Madame Lulu, From LAPL, HH-3E Jolly Green Giant, HH-53 Super Jolly Green Giants, IRN Ayatollah Ruhollah Khomeini, IRN Iran, KHM Cambodia, KHM Cambodian Civil War (1967-1975), KHM Khmer Rouge, KHM King & President Norodom Sihanouk, KHM Phnom Penh, KHM Tonle Sap (Great Lake), LAO Attopeu, LAO Ban Ban, LAO Ban Ban Valley, LAO Ban Son, LAO Bataillon Guerrier 403 (Laotian Civil War), LAO Blind Bonze Pho Satheu, LAO Bolovens Plateau, LAO Colonel Deuan Sunnalath, LAO Communist Neutralists, LAO Defense Minister Sisouk Na Champassak, LAO Hmong Meo Tribesmen, LAO Hotel Lima, LAO ICC Internationl Control Commission, LAO Ice House One and Two, LAO Jungle's Mouth, LAO Khang Khay, LAO Khang Khay - Chinese Cultural Center, LAO King Savang Vatthana, LAO King Sisavang Vong, LAO Klick 11, LAO Lam Son 719 (1971) (Vietnam War), LAO Lan Xiang 9 - Raven Hooch, LAO Laos, LAO Laotian Civil War (1959-1975), LAO Les Rendezvous des Amis, LAO Lhat Houang, LAO Lima 35 - Paksane (Laotian Civil War), LAO Lima Site 103 - Phu Da Pho (Laotian Civil War), LAO Lima Site 108 - Moung Soui (Laotian Civil War), LAO Lima Site 113 - Moung Cha (Laotian Civil War), LAO Lima Site 15 - Ba Na (Laotian Civil War), LAO Lima Site 15 - Phong Saly (Laotian Civil War), LAO Lima Site 20 - Sam Thong (Laotian Civil War), LAO Lima Site 204 - Khang Kho (Laotian Civil War), LAO Lima Site 20A - Long Tieng (Laotian Civil War), LAO Lima Site 262 - Ban Xon / Ban Houei Pamone (Laotian Civil War), LAO Lima Site 276 - Lat Sen (Laotian Civil War), LAO Lima Site 32 - Boung Lam (Laotian Civil War), LAO Lima Site 36 - Na Khang (Laotian Civil War), LAO Lima Site 46 - Seno (Laotian Civil War), LAO Lima Site 85 - Phu Pha Thi (Laotian Civil War), LAO Long Tieng, LAO Luang Prabang, LAO Moung Soui, LAO MR Military Region (Laotian Civil War), LAO MR1 (Laotian Civil War), LAO MR2 (Laotian Civil War), LAO MR3 (Laotian Civil War), LAO MR4 (Laotian Civil War), LAO MR5 (Laotian Civil War), LAO Muong Mai, LAO Nong Het, LAO Operation About Face / Kou Kiet (1969) (Laotian Civil War), LAO Operation Barrel Roll (1964-1973) (Laotian Civil War) (Vietnam War), LAO Operation Bedrock (1971) (Laotian Civil War), LAO Operation Black Lion IV (1972) (Laotian Civil War), LAO Operation Blind Bat (1964-1970) (Laotian Civil War), LAO Operation Glass A (Laotian Civil War), LAO Operation Goodlook (1970) (Laotian Civil War), LAO Operation Leapfrog (1970) (Laotian Civil War), LAO Operation Nighty-Night (1969) (Laotian Civil War), LAO Operation Northwind (Laotian Civil War), LAO Operation Pig Fat (1968) (Laotian Civil War), LAO Operation Rain Dance (1969) (Laotian Civil War), LAO Operation Shining Brass / Prairie Fire / Phu Dong (1965-1975) (Laotian Civil War) (Vietnam War), LAO Operation Steel Tiger (1965-1968) (Laotian Civil War) (Vietnam War), LAO Operation Tiger Hound (1965-1968) (Laotian Civil War) (Vietnam War), LAO Operation Westwind (Laotian Civil War), LAO Operation White Star (1959-1961) (Laotian Civil War), LAO Operation X (1946-1954) (French Indochina War), LAO Operation Yankee Team (1964) (Laotian Civil War), LAO Padong, LAO Pakse, LAO Paksong, LAO Pathet Lao, LAO Phong Saly, LAO Phou Khean, LAO Phou Nok Kok (Black Lion), LAO Phou Tham, LAO Plain of Jars / Plaine des Jarres, LAO Prime Minister Phoui Sananikone, LAO Prime Minister Souvanna Phouma, LAO Prince Souvanna Phouma, LAO Project Waterpump (1964-1973) (Laotian Civil War), LAO Purple Porpoise, LAO RLA Captain Kong Le, LAO RLA General Oudone Sananikone, LAO RLA General Phoumi Nosavan, LAO RLA General Vang Pao, LAO RLA Royal Laotian Army, LAO RLA SGU Special Guerrilla Units, LAO RLAF CPK Chao Pha Khao Hmong Pilots/Backseaters (Laotian Civil War), LAO RLAF Lt Colonel Lee Lue, LAO RLAF Royal Lao Air Force, LAO Roadrunner Lake, LAO Route 13, LAO Route 19, LAO Route 23, LAO Route 4, LAO Route 6, LAO Route 7, LAO Route 7/71 Junction, LAO Route 71, LAO Sam Nuea, LAO Sam Thong, LAO Saravane, LAO Se Kong River, LAO Skyline Ridge, LAO St. Valentine's Day Massacre (Laotian Civil War), LAO Tchepone, LAO US Programs Evaluation Office (Laotian Civil War), LAO USAF Butterfly FAC (Laotian Civil War), LAO USAF Project 404 (Laotian Civil War), LAO USAF Steve Canyon Program - Ravens FAC (Laotian Civil War), LAO Vientiane, LAO Vientiane - US Air Attache (Laotian Civil War), LAO Vientiane - US Embassy (Laotian Civil War), LAO Wattay Airport, LAO White Rose, LAO Xieng Khouang, LBY Colonel Muammar al-Qaddafi, LBY Libya, O-1 Bird Dog, OV-10 Bronco, Pilatus Porter, PT-76 Amphibious Light Tank, SpecOps, T-28 Trojan, THA Ban Vinai, THA CIA 4802 Joint Liaison Detachment Logistics Office - Udorn (Laotian Civil War), THA Nam Phang, THA PARU Border Patrol Police Aerial Resupply Unit, THA RTAF Royal Thai Air Force, THA RTAFB Nakhon Phanom Royal Thai Air Base, THA RTAFB Ubon Royal Thai Air Base, THA RTAFB Udorn Royal Thai Air Base, THA Thailand, THA USAF ISC Infiltration Surveillance Center - Nakhon Phanom (Igloo White) (Vietnam War), U-17 Skywagon, US Air America Eugene Hasenfus, US Albert Hakim, US Ambassador George McMurtrie Godley III, US Ambassador Llewellyn Thompson, US Ambassador William Sullivan, US Averrell Harriman (Governor of NY) (Ambassador at Large), US CIA Anthony Posepny (Tony Poe), US CIA Burr Smith, US CIA Central Intelligence Agency, US CIA Dave Morales, US CIA Douglas Blaufarb, US CIA Ed Wilson, US CIA Frank Snepp, US CIA Henry Hecksher, US CIA Jerry 'Hog' Daniels, US CIA John Stockwell, US CIA Kham Sing (Gold Lion), US CIA Lawrence Devlin, US CIA Pat Landry, US CIA Phillip Agee, US CIA Richard Helms, US CIA Ted Shackley, US CIA Tom Clines, US CIA Will Green (Black Lion), US CIA William Colby, US COA CASI Continental Air Services International, US COA Continental Airlines, US Dr. Henry Kissinger, US Edgar "Pop" Buell, US Father Lucien Bouchard (Catholic Priest), US Iran-Conta Affair, US John Gunther Dean (Diplomat), US President Dwight D. Eisenhower, US Secretary of Defense Melvin Laird, US Secretary of State Dean Rusk, US Senator Edward Kennedy, US Senator J. William Fullbright, US Senator Stuart Symington, US State Department, US T.D. Allman (Journalist), US Tom Corcoran (Diplomat), US USA Biological Warfare Corps, US USA General Earle Wheeler, US USA General William Westmoreland, US USA Green Berets, US USA United States Army, US USA USSF Special Forces, US USAF 14th Air Commando Wing, US USAF 1st Air Commando Wing, US USAF 1st Air Commando Wing - Det 6 (Laotian Civil War), US USAF 20th Helicopter Squadron, US USAF 22nd Special Operations Sqd - Zorro, US USAF 23rd TASS - NAIL FAC, US USAF 23rd TASS - OL1 - Rustic FAC (Ubon) (Cambodian Civil War) (Vietnam War), US USAF 56th Air Commando Wing, US USAF 56th Special Operations Wing, US USAF 56th Special Operations Wing - Det 1, US USAF 7th ABCCC Airborne Command and Control Sqd - Cricket, US USAF 7th Air Force, US USAF 7th/13th Air Force, US USAF Colonel Mike Heenan, US USAF Fairchild Air Force Base WA, US USAF General Curtis LeMay, US USAF General George Brown, US USAF General John D. Lavelle, US USAF General Louis T. Seith, US USAF General Richard V. Secord, US USAF General William Momyer, US USAF Generl Robert L. Petit, US USAF Gus Sonnenberg, US USAF Hurlburt Field FL, US USAF JEST Jungle Environment Survival Training, US USAF Lt Colonel George Vogel, US USAF Lt Colonel Mark Berent, US USAF Major General Harry Heinie Aderholt, US USAF Major John Clark Pratt, US USAF United States Air Force, US USMC Lt Colonel Oliver North, US USMC United States Marine Corps, USAID, USSR 1st Secretary Nikita Khrushchev, USSR Foreign Minister Andrei Gromyko, VNM 1972 Easter Offensive (1972) (Vietnam War), VNM Bien Hoa, VNM Bien Hoa Air Base (Vietnam War), VNM CIA Air America (1950-1976) (Vietnam War), VNM DRV Lao Dong Party, VNM DRV NVA 148th Regiment, VNM DRV NVA 174th Regiment, VNM DRV NVA 312th Division, VNM DRV NVA 316th Division, VNM DRV NVA 766th Regiment, VNM DRV NVA General Vo Nguyen Giap, VNM DRV NVA Group 559, VNM DRV NVA North Vietnamese Army, VNM DRV NVAF North Vietnamese Air Force, VNM DRV VC Viet Cong, VNM DRV VM 304th Division, VNM DRV VM Regiment 98, VNM DRV VM Viet Minh, VNM FRA ADT Cap St Jacques Military School, VNM French Indochina War (1946-1954), VNM Green Beret Affair (Vietnam War), VNM Ho Chi Minh Trail (Vietnam War), VNM II Corps (Vietnam War), VNM Nha Trang, VNM Operation Arc Light (1965-1973) (Vietnam War), VNM Operation Igloo White (1968-1973) (Vietnam War), VNM Operation Linebacker II (1972) (Vietnam War), VNM Operation Pony Express (1965-1969) (Laotian Civil War) (Cambodian Civil War) (Vietnam War), VNM Paris Peace Accords (1973) (Vietnam War), VNM Phan Rang Air Base, VNM RVN ARVN Army of the Republic of Vietnam, VNM RVN Nguyen Van Thieu, VNM RVN Vietnamization Policy (Vietnam War), VNM US MACV Military Assistance Command Vietnam (Vietnam War), VNM USAF TACC Tactical Air Control Center - BLUE CHIP (Vietnam War), VNM Vietnam, VNM Vietnam War (1955-1975) Rating: ★★★★★ (5 Stars) Subject: Books.Military.20th-21st Century.Asia.Vietnam War.Laotian Civil War.Aviation.FAC.Ravens

Description: Officially the war in Laos did not exist - both North Vietnam and the USA denied they had troops there. In fact, thousands of North Vietnamese were invading the country and pouring down the Ho Chi Minh Trail on their way to the south, and the Americans were fighting a vigorous war against them from the air.

The Ravens were the pilots, all volunteers, who flew through heavy groundfire to identify targets and call in air-strikes. Their mission was so secret that they were 'sold' their prop-driven planes for a dollar apiece so they could be struck from US Air Force records. They wore no uniform and carried no identification. Refugees from the bureaucracy of the war in Vietnam, they accepted the murderous casualty rates of what was known as the Steve Canyon Program in return for a life of unrestricted flying and fighting.

Devoted to the hill tribesmen they fought alongside, the Ravens did their job with extraordinary skill and crazy courage and with a humour that was all of its own. This is the story, brilliantly told for the first time, of these extraordinary men. Based on extensive interviews with the survivors, it is a tale of undeniable heroism, blending real-life romance, adventure and tragedy.

Review: This was a great book with a lot of problems. 

The #1 problem was that I was reading an ebook version from Apostrophe books that was, quite simply, poorly done. So many issues with the conversion... I’s became 1′s... places and names were spelled 3 different ways throughout the book... issues like that.

The #2 problem was that there were multiple stories being told in a book about one story. This book had some good info on the Raven FACs, but it also went deeply into the story of the Laotian Civil War, the French Indochina War, the Hmong people, Henry Kissinger and the Nixon Administration. All of these things are intertwined and important, but the author uses up a LOT of the books real estate for these topics which tends to take a lot of the focus away form the Ravens, which the book is supposed to be about. 

It’s still a 5 star book because it does go deeply into the stories of the who, the what, the when, and the where. You get a real good feel for the cast of characters that made up the Raven FACs, and you learn a lot about the Laotian Civil War, the CIA, the political issues and more. 

It’s really a good primer and a good way to get a feel for what happened in the region. 

[monstersdownthepath]

Juiblex(3e)/Jubilex(PF) has always been my favorite of the Demon Lords/Princes, so I'm super happy that not only does Jubilex have a very easy Obedience (which we'll cover on Monday because I'm a liar and I can't stop talking), but Boons that are flat-out fantastic as well.

It's a shame that Pathfinder didn't steal a page from 3.5s works and make Thrall of X classes, though. In 3e, there were prestige classes for demon worshipers called Thrall of X, where X was the name of a Demon Prince. Among them was the Thrall of Juiblex, a class poorly balanced (to the point it doesn't even seem proofread) in the best sense of the word.

Full BAB, three good saves, and a handful of interesting and powerful abilities that made for a decent melee character, such as an acidic touch attack, Contagion once a day, and an aura that sickened nearby enemies. It was made all the better by their ability to summon handfuls of demons once a day... and as many slimes and oozes as they felt like, constantly, without limit.

Yes, while their Summon Demons ability was only usable once a day, a Thrall of Juiblex 3 could Summon Ooze without any limits or restrictions beyond the fact they could only conjure Green Slime, Ochre Jelly, or a Gelatinous Cube. All three of those have their uses even at high level, and there are very few creatures that can deal with an endless wave of slime. It's even better once the Thrall reaches level 7, because then you can summon Black Pudding as often as you feel like.

Obviously, those abilities require a per-day limit before a DM would even think of allowing them, but even with such a limit a Thrall can still pull their weight. At level 4 they can use Alter Self at will to throw on whatever face they may need... and at level 9, can use Polymorph Self at will to throw on whatever face, size category, natural attacks, or movement modes it needs. In 3.5, Polymorph healed the targets, so a Thrall could restore it's own HP just by shifting itself constantly while out of combat.

I love this class. I love this slime demon.

[alapporte19ahs]

The 3 Ps Assessment: Parties, Political Interest Groups, and PACs

1a. Republican: Restrictions on energy is taking thousands of jobs and hurting the American economy. We nee to use our own oil, “clean coal”, natural gas, solar, geothermal, and nuclear energy to relieve our dependence on Middle east oil.

Democrat: America should be running on clean energy by the mid-centry. American states are already seeing an increase in hurricanes, droughts, and flooding. The need for clean energy means there’s a need for new jobs to work for clean energy companies instead of big oil.

Libertarian: Governments shouldn’t be subsidizing any form of energy and they have a terrible track record with the environment, so they aren’t responsible for it. We shouldn’t trust governments with out environment and energy resources.

Green: Climate change is the largest threat facing America and the world today. They want to cut 95% of greenhouse gas emissions by 2050. They want to encourage and create clean energy jobs. They want a strong international climate treaty. They want an economic policy for a safer climate treaty. They want clean agriculture.

Peace and freedom:They believe that socialism is necessary to end the destructiveness of capitalism on our environment. They want to out-law unsustainable practices such as clear cutting and fracking. They want free public transportation, and to eliminate nuclear power plants. They want to restore the damage in our air and water systems. They also want to end environmental racism.

1b. Republican: I agree that we need to relieve our dependence on oil from the Middle East, but we don’t have the same reserves as them. We don’t have enough oil in the U.S to support how much we use. We need to be relieving dependence on all oil, natural gas, and coal. And focus on solar energy.

Democrat: I agree with almost everything the democratic party said about climate change. But their predictions aren’t realistic. Scientists say we’re not doing nearly enough to keep “global temperature increases to “well below” two degrees Celsius and to pursue efforts to limit global temperature increases to 1.5 degrees Celsius.”(as agreed in the Paris agreement). To do this we need to lower our meat consumption greatly, but it doesn’t state that as ways we can lower our CO2 and methane emissions.

Libertarian:I agree the the government has a bad tech record with the environment, but in dire times the government should take control and restrict certain things to ensure public safety and health for generations to come.

Green: I agree with all of the libertarians points. I don’t know that they’re reachable, but they do include the effects of animal agriculture. they want to encourage less meat consumption, and promote organic and local food production and distribution.

Peace and freedom: I agree with everything that peace and freedom says. Sadly, this is the only way we can really stop climate change, and ensure life on earth for the human race.

1c. I agreed most with the Peace and Freedom’s platform. This doesn’t surprise me too much because although they're views are extreme, we need an extreme and radical change to combat climate change.

2a. Sierra Club

2b. "The purposes of the Sierra Club are to explore, enjoy, and protect the wild places of the earth; to practice and promote the responsible use of the earth's ecosystems and resources; to educate and enlist humanity to protect and restore the quality of the natural and human environment; and to use all lawful means to carry out these objectives."

2c. 1. Protect wild places and endangered species

      2. Keep our air and water clean

      3. Ensure a clean energy future

      4. Curb climate change

      5. Keep the pressure on politicians and corporations to ensure safe and healthy communities

2d. The Sierra Club endorses the legislation 1631 that “holds polluters accountable by investing in projects that will lead to cleaner air, cleaner water, and a healthier future for Washington.”

2e. There are Sierra Clubs located around the nation, but there are local ones in Oakland and Berkely. On October 27, there’s a “ walk through the city of the dead” at the Colma BART station. On October 29 there’s a “speak up for SF’s resolution supporting the bay-delta protection plan” in San Francisco city hall. There’s the “West Contra Costa monthly group meeting” on November 3 in Berkely.

2f. Yes there are volunteer opportunities including planting trees, canvassing, and attending hikes.

3a. California Environmental Justice Alliance 

3b.  "The California Environmental Justice Alliance is a statewide, community-led alliance that works to achieve environmental justice by advancing policy solutions. We unite the powerful local organizing of our members in the communities most impacted by environmental hazards- low-income communities and communities of color- to create comprehensive opportunities for change at a statewide level. We build the power of communities across California to create policies that will alleviate poverty and pollution. Together, we are growing the statewide movement for environmental health and social justice."

3c.1. Replacing big polluting industries with a green, locally based and sustainable economy 

     2. People’s lives and health are more important than big business profit

     3. Working to have no barriers to opportunity for low-income communities and communities of color.

     4. Working to have no discrimination in state policies and practices.

     5. Working to relay only on clean energy and eliminate toxic industries that pollute our water, land, and health.

3d. The CEJA endorses AB 523  by Assemblymember Reyes to expand access to clean energy in California.This will help break down barriers that don’t allow low income  communities to have affordable access to clean energy.

3e. They’re located on Oakland. I did not find any local meeting I could attend.

3f. Yes you can volunteer to send a pre-written email to governor Brown asking him sign AB 523.

4. Both the Sierra Club and the CEJA seemed pretty organized, but the Sierra club had more opportunities to get involved and just generally more to explore on their site. The sierra Club seems more successful, partially because they have more ways to get involved on their website. The Sierra Club targets people around the nation that are concerned about the environment and want to get involved, while the CEJA is geared toward Californian’s who are concerned about the environment. The CEJA seemed to project more concern for the health of low-income areas, and people of color that are often not represented in environmental justice, and are at the highest health risks because of where they live and the amount of money the have (environmental racism). The CEJA’s supporters include APEN (Asian Pacific Environmental Network), CBE (Communities for a better Environment), CCAEJ ( Center for Community Action and Environmental Justice), and CAUSE ( Central Coast Alliance United for a Sustainable Economy).

5a. Koch Industries

5b. “We challenge ourselves to improve people’s lives by creating better products using fewer resources. At the same time, we challenge barriers that hinder competition, opportunity, innovation and progress.”

5c. Total receipt: $3,767,334

     Spent: $3,754,267

     cash on hand: $1,602,378

5d. they have given a total of $1,488,000 to candidates. 2% went to democrats, and 98% went to republicans.

5e. Some of their donors include Koch Industries, Invista Sarl, and Molex LLC. Invista makes chemicals, polymers, fabrics and fibers for clothing, carpets, and car parts. Molex LLC is a technology company that makes products such as lighting, and cables. Most of their donations come from their own company.

[generalalmondbagelflower]

Check out this listing I just added to my Poshmark closet: FREE PEOPLE MOVEMENT Blue/ Green Color joggers.

[otowery18ahsgov-blog]

Blog Post #7 The 3 Ps Assessment: Parties, Political Interest Groups, and PACs

1a. Republicans, Democrats, Libertarian, Green and Peace and Freedom all support equal rights for women in the workplace. When reproductive services like abortion come into play, Republicans are not supportive of equal rights for women.

1b. I believe that women should have equal rights in the workplace and frankly all things outside the workplace as well. I agree with any party that supports that.

1c. In terms of women’s rights I think I agree most with the Democrat party because of the advocates I’ve seen speak about the issue. I would not consider myself a full Democrat when it comes to other aspects of the party.

2a. National Organization for Women

2b. National Organization for Women's goal has been to take action to bring about equality for all women. NOW works to eliminate discrimination and harassment in the workplace, schools, the justice system, and all other sectors of society; secure abortion, birth control and reproductive rights for all women; end all forms of violence against women; eradicate racism, sexism and homophobia; and promote equality and justice in our society

2c. They are the largest feminist organization in the US, the litigation efforts of the Foundation seek to protect reproductive health options, NOW engages in voter empowerment programs designed to encourage women to register, vote, and stay politically active in their communities, launched the Love Your Body Campaign aimed at young women to promote positive body image outlooks, and NOW Foundation has been involved extensively in preparing reports about how the Social Security program can be updated to help women as caregivers as well as improve the retirement security of women with disabilities, women of color and lifetime low-income earners.

2d. NOW supports the Voting Rights Advancement Act that will afford protections to ensure that all members of our society can exercise their fundamental right to vote.

2e. NOW is located in D.C but has chapters all over the country. The CA NOW chapter has a conference in July in San Jose.

2f. Internships are available.

2g. It was founded in 1966.

3a. National Women’s Political Caucus of California

3b. NWPC CA recruits, trains and supports pro-choice women candidates who support our “bottom line issues” for elected and appointed offices at all levels of government regardless of party affiliation.

3c. The Caucus offers campaign training for candidates and campaign managers, endorses strong female congresswomen and senators, advocate for more women in office, committed to choice and publicly funded abortion, and NWPC CA supports meaningful preventive training programs for employers and workers.

3d. They are supporting Governor candidate Delaine Eastin.

3e. They are located in Tustin, CA but there is a caucus in the Silicon Valley.

3f. Internships are available.

3g. NWPC was founded in the midst of the modern American women’s movement when 320 women from 26 states met under the leadership of feminists such as Shirley Chisholm, Bella Abzug, Gloria Steinem, and Betty Friedan to form the caucus on July 11, 1971.

4. The NOW seems more organized and successful because it seems to have the largest involvement. They both target women and support women’s rights but NWPC is heavily focused on getting more women in political positions.

5a. PAC name: Emily’s List

5b. Works for larger leadership roles for pro-choice Democratic women in legislative bodies and executive seats so that families can benefit from the open-minded, productive contributions that women have consistently made in office.

5c. They have made $40,710,828, spent $35,245,689, and have $10,849,323 in cash on hand.

5d. Spent all on Democrats, none on Republicans.

5e. Some of their donors are the Kate Brown Committee, Eve Rice, and Brian Eaton (Google). I have no idea who these people are but I found a handful of donors who were unemployed but still donated a lot of money, which I thought was interesting. They must really care and believe in Emily’s List’s mission. 

[fic-dreamin]

Nice concept lousy execution. I've been a fan of superheroes as far back as I can remember. My first RPG was the basic set for Go to Amazon

A very good game, but not for beginners The game is good, it is well written and has only a few small errors (there is an errata sheet correcting those out there somewhere. However, the powers section requires a bit of work if you want to do anything past the basics. Unlike previous versions of Mutants and Masterminds (upon which this is based), this version mainly gives power effects and expects the player to design their powers using those. This requires imagination, the ability to calculate things, and a bit of maturity not to get out of hand. It is very good for experienced players who can handle it, but is probably not the best game for starting to learn how to play. Go to Amazon

Arrived in great condition, through and perfect for any RP-er I recently started DM-ing a superhero-themed game, and let me tell you, even if you're not using any DC heroes; this is a fantastic world-building and point-system guide. It includes virtually any power you'd ever want to include in game, as well as drawbacks and limits that are necessary for a power-themed session to go smoothly. It has a great way of breaking down powers you can have as a super into "abilities, powers, and skills" categories, which are great for distinction between skill heroes like Nightwing vs. power-heavy ones like Superman. AND it has a glossary of several popular hero sheets, so you can go toe-to-toe with Batman if you're feeling saucy. Go to Amazon

DC Adventures (M&M 3E) Perhaps the best Super RPG in history. This is one of the best systems I have ever read. Second only to the original DC Heroes, which depicts comic book heroes a bit better, but does not hold up as well when dealing with heroes of various power levels. Batman in DC Adventures can hang with Superman more easily than he can in DC Heroes. I thought the second edition of M&M was just as good as the Hero System. The Hero System was perhaps more thorough, but you could make anything with M&M 2E you could make with Hero, and the simplicity streamlined rules of M&M 2E more than made up for thoroughness of the much more complicated system that Hero is. To be honest, I don't find Hero that complicated, though you can definitely see how it is, when introducing it to a new set of players. When I read DC Adventures using M&M 3E, I wasn't sure it could beat M&M 2E, but it more than beat it, it fixed nearly all the problems I had with 2E. Some optional rules from 3E Game Masters Guide and adding in a few easily adaptable optional rules from 2E's Mastermind's Manual (namely the tactical movement, and perhaps the damage roll) and the only thing I have heard any complaints about is the damage system. (And that is only from one person who likes to roll damage instead of resisting it. Again, the damage roll variant from M&M 2E MM may even fix this. Go to Amazon

Nicely done I can't really argue with previous reviews here, and would add that the layout confusion in this book isn't really "fixed" in the actual M&M 3E book either - this looks to be laid out almost the same way but with DC artwork and examples, and a slightly more polished appearance. Go to Amazon

Neat but complicated Love the idea, but the combat system is just too complicated and it makes it a little hard to have fun with it. It just takes too long to learn. Call me a casual, but after studying it extensively and still having trouble figuring out certain mechanics during game play really hampers fun. Go to Amazon

A good game for players and GMs who enjoy system mastery. Green Ronin did a great job of translating the d20 mechanic into a system ... Not just the best DC roleplaying game-- one of the best RPG's ever! I wouldn't not recommend it. Five Stars A Must Have Fantastic Book Five Stars Strong addition I Liked This Book So Much, I Bought Many More Fantastic!!!!

[poetryofchrist]

Leviticus 2

What to do with handful, fistful. What is this extraction from the oblation. Remember also that rich word קרב, (do a find ctrl-f to see how often the first two letters occur in this chapter). קרב 'means' one of: approach (69) bring near (95) bring together (1) brought near (27) came near (12) cheek jowl (3) close (1) close combat (18) come near (9) coming near (1) draw near (6) entrails (21) far concern (1) inner (1) near (53) near-term (1) nearby (1) nearness (3) oblation (80) quick (1) quite near (4) [these last only for acrostic q's]. So fistful / handful - extraction. Is it a full pinch? I have used pinch in Job and Proverbs - too bad. Along with wink, they are now fixed in the mysteries of the sand. I don't like to break my rules for specifics. I am happy to let them lapse (with control) for common generic movements, like come, go, bring etc. So what we have is a generous extraction from the oblation sufficient for smoke and fire. At a minimum, the ritual of the offering says: don't think you know everything and have everything under control. Things are subject to such awareness of what is unknown to us. Es-que c'est toi, mon amour?

Leviticus 2 Fn Min Max Syll וְנֶ֗פֶשׁ כִּֽי־תַקְרִ֞יב קָרְבַּ֤ן מִנְחָה֙ לַֽיהוָ֔ה סֹ֖לֶת יִהְיֶ֣ה קָרְבָּנ֑וֹ וְיָצַ֤ק עָלֶ֙יהָ֙ שֶׁ֔מֶן וְנָתַ֥ן עָלֶ֖יהָ לְבֹנָֽה 1 And the self that will bring near an oblation, a gift for Yahweh, his oblation will be fine flour, and he will infuse on it oil and daub on it frankincense. 3e 4C 19 17 וֶֽהֱבִיאָ֗הּ אֶל־בְּנֵ֣י אַהֲרֹן֮ הַכֹּהֲנִים֒ וְקָמַ֨ץ מִשָּׁ֜ם מְלֹ֣א קֻמְצ֗וֹ מִסָּלְתָּהּ֙ וּמִשַּׁמְנָ֔הּ עַ֖ל כָּל־לְבֹנָתָ֑הּ וְהִקְטִ֨יר הַכֹּהֵ֜ן אֶת־אַזְכָּרָתָהּ֙ הַמִּזְבֵּ֔חָה אִשֵּׁ֛ה רֵ֥יחַ נִיחֹ֖חַ לַיהוָֽה 2 And he will bring it to the children of Aaron the priests, and he will extract from it a full extraction of its fine flour and its oil with all its frankincense, and the priest will make its memorial burn with smoke toward the altar, an offering by fire, a restful fragrance for Yahweh. 3d 4B 36 24 וְהַנּוֹתֶ֙רֶת֙ מִן־הַמִּנְחָ֔ה לְאַהֲרֹ֖ן וּלְבָנָ֑יו קֹ֥דֶשׁ קָֽדָשִׁ֖ים מֵאִשֵּׁ֥י יְהוָֽה 3 And the remains from the gift is for Aaron and for his children, holy of holy things from the fire of Yahweh. 3e 4A 16 10 וְכִ֥י תַקְרִ֛ב קָרְבַּ֥ן מִנְחָ֖ה מַאֲפֵ֣ה תַנּ֑וּר סֹ֣לֶת חַלּ֤וֹת מַצֹּת֙ בְּלוּלֹ֣ת בַּשֶּׁ֔מֶן וּרְקִיקֵ֥י מַצּ֖וֹת מְשֻׁחִ֥ים בַּשָּֽׁמֶן 4 And if you approach with an oblation, a gift baked in an oven, fine flour cakes, unleavened, mingled with oil and wafers of unleavened bread, anointed with oil. 3d 4C 13 23 וְאִם־מִנְחָ֥ה עַל־הַֽמַּחֲבַ֖ת קָרְבָּנֶ֑ךָ סֹ֛לֶת בְּלוּלָ֥ה בַשֶּׁ֖מֶן מַצָּ֥ה תִהְיֶֽה 5 And if your oblation is a gift on the pan, fine flour mingled with oil, unleavened bread, this will be. 3d 4A 13 12 פָּת֤וֹת אֹתָהּ֙ פִּתִּ֔ים וְיָצַקְתָּ֥ עָלֶ֖יהָ שָׁ֑מֶן מִנְחָ֖ה הִֽוא 6 Fragment it into fragments and infuse it with oil. A gift it is. 3e 4C 15 3 וְאִם־מִנְחַ֥ת מַרְחֶ֖שֶׁת קָרְבָּנֶ֑ךָ סֹ֥לֶת בַּשֶּׁ֖מֶן תֵּעָשֶֽׂה 7 And if your oblation is a gift stirred, fine flour with oil you will make. 3e 4A 11 8 וְהֵבֵאתָ֣ אֶת־הַמִּנְחָ֗ה אֲשֶׁ֧ר יֵעָשֶׂ֛ה מֵאֵ֖לֶּה לַיהוָ֑ה וְהִקְרִיבָהּ֙ אֶל־הַכֹּהֵ֔ן וְהִגִּישָׁ֖הּ אֶל־הַמִּזְבֵּֽחַ 8 And you will bring the gift that is made from these for Yahweh, and when it is brought near to the priest, then he will come close to the altar. 3c 4B 18 17 וְהֵרִ֨ים הַכֹּהֵ֤ן מִן־הַמִּנְחָה֙ אֶת־אַזְכָּ֣רָתָ֔הּ וְהִקְטִ֖יר הַמִּזְבֵּ֑חָה אִשֵּׁ֛ה רֵ֥יחַ נִיחֹ֖חַ לַיהוָֽה 9 And the priest will contribute from the gift its memorial and will make it burn with smoke toward the altar, an offering by fire, a restful fragrance for Yahweh. 3d 4C 22 9 וְהַנּוֹתֶ֙רֶת֙ מִן־הַמִּנְחָ֔ה לְאַהֲרֹ֖ן וּלְבָנָ֑יו קֹ֥דֶשׁ קָֽדָשִׁ֖ים מֵאִשֵּׁ֥י יְהוָֽה 10 And the remains from the gift is for Aaron and for his children, holy of holy things from the fire of Yahweh. 3e 4A 16 10 כָּל־הַמִּנְחָ֗ה אֲשֶׁ֤ר תַּקְרִ֙יבוּ֙ לַיהוָ֔ה לֹ֥א תֵעָשֶׂ֖ה חָמֵ֑ץ כִּ֤י כָל־שְׂאֹר֙ וְכָל־דְּבַ֔שׁ לֹֽא־תַקְטִ֧ירוּ מִמֶּ֛נּוּ אִשֶּׁ֖ה לַֽיהוָֽה 11 Every gift that you will bring near for Yahweh you will not make with leaven, because all yeast and all honey you will not make it burn with smoke for an offering by fire to Yahweh. 3c 4C 17 19 קָרְבַּ֥ן רֵאשִׁ֛ית תַּקְרִ֥יבוּ אֹתָ֖ם לַיהוָ֑ה וְאֶל־הַמִּזְבֵּ֥חַ לֹא־יַעֲל֖וּ לְרֵ֥יחַ נִיחֹֽחַ 12 As an oblation of first-fruits, you may bring them near Yahweh, but on the altar they will not be offered for a restful fragrance. 3d 4A 11 15 וְכָל־קָרְבַּ֣ן מִנְחָתְךָ֮ בַּמֶּ֣לַח תִּמְלָח֒ וְלֹ֣א תַשְׁבִּ֗ית מֶ֚לַח בְּרִ֣ית אֱלֹהֶ֔יךָ מֵעַ֖ל מִנְחָתֶ֑ךָ עַ֥ל כָּל־קָרְבָּנְךָ֖ תַּקְרִ֥יב מֶֽלַח 13 And every oblation of your gifts with salt you will salt, and you will not cease to salt the covenant of your God from your gifts. Over all your oblations, you will come near with salt. 3e 4C 30 9 וְאִם־תַּקְרִ֛יב מִנְחַ֥ת בִּכּוּרִ֖ים לַיהוָ֑ה אָבִ֞יב קָל֤וּי בָּאֵשׁ֙ גֶּ֣רֶשׂ כַּרְמֶ֔ל תַּקְרִ֕יב אֵ֖ת מִנְחַ֥ת בִּכּוּרֶֽיךָ 14 And if you bring near the gift of first ripe fruit to Yahweh, green shoots of parched grain in the fire, plantation beaten corn, you will bring near for the gift of your first ripe fruit. 3d 4C 11 19 וְנָתַתָּ֤ עָלֶ֙יהָ֙ שֶׁ֔מֶן וְשַׂמְתָּ֥ עָלֶ֖יהָ לְבֹנָ֑ה מִנְחָ֖ה הִֽוא 15 And you will give it with oil and put frankincense on it. A gift it is. 3e 4C 18 3 וְהִקְטִ֨יר הַכֹּהֵ֜ן אֶת־אַזְכָּרָתָ֗הּ מִגִּרְשָׂהּ֙ וּמִשַּׁמְנָ֔הּ עַ֖ל כָּל־לְבֹנָתָ֑הּ אִשֶּׁ֖ה לַיהוָֽה 16 And the priest will make its memorial burn with smoke from the beaten corn and its oil with all its frankincense, an offering by fire to Yahweh. 3e 4A 24 4

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[naivelocus]

Protein expression guided chemical profiling of living cells by the simultaneous observation of Raman scattering and anti-Stokes fluorescence emission

Anti-Stokes fluorescence excitation of fluorescent proteins

Despite several scattered reports on the anti-Stokes fluorescence emission from chloroplasts25, organic26,27 and inorganic dyes28, to the best of our knowledge, the anti-Stokes fluorescence excitation of FPs has never been reported before. To verify whether the anti-Stokes emission from FPs can be detected under Raman measuring conditions, we recorded the emission spectra of various FPs with a shorter emission peak than the continuous-wave (cw) 532 nm laser that we use for our Raman experiments (Fig. 2a). For the FPs with emission spectra much shorter than 532 nm, i.e. blue fluorescent protein (BFP, emission peak at 445 nm), almost no fluorescence signals can be detected. However, for FPs with a closer emission spectra to 532 nm, i.e. enhanced cyan fluorescent protein (ECFP, emission peak at 476 nm), monomer teal fluorescent protein (mTFP, emission peak at 492 nm) and green fluorescent protein (GFP, emission peak at 509 nm), an obvious fluorescence emission peak can be seen at the anti-Stokes side of the spectra. One point that is worth mentioning is the absence of resonance enhanced Raman peaks of the fluorescent protein chromophore at the Stokes region of the spectra. This proves that our anti-Stokes fluorescence excitation method will introduce minimum perturbation to the Stokes spectral window that is left for Raman measurements. Note that in Fig. 2a, the GFP spectrum has its intensity divided by 100 times, which means that it has a much stronger emission than all other FPs. The anti-Stokes fluorescence excitation process is a universal phenomenon that is not limited by the combination of excitation laser and FPs mentioned above. As long as the absorption peak of the FPs is within a certain range to the excitation laser, there is a huge flexibility for the choice of excitation wavelengths and FPs (Sup. 1). These facts already suggest that the fluorescing mechanism we report here is not a multiphoton excitation scheme of the ultraviolet absorption peak of FPs as one of our previous reports indicated29.

Figure 2

(a) The anti-Stokes fluorescence emission spectra of BFP, ECFP, mTFP and GFP. The spectral valley around 532 nm is due to the notch filter, and the weak remaining of Rayleigh scattering at 532 nm can still be seen at the centre of the spectral valley. Note that the excitation intensity for GFP is 100 times lower than the other FPs, because the emission spectrum for GFP would saturate otherwise. (b,c) shows the fluorescence response according to excitation intensity of ECFP and mTFP. Both of them shows a slope at around 1 in the log plot, which indicates the anti-Stokes excitation of FPs is a single-photon process. Standard deviation bars of each spot are from 3 repeated measurements. The blue region between 1~4 mW/μm2 excitation intensity is the common condition to measure the Raman spectra of cells.

To further identify the fluorescing mechanism of the FPs, we verified their fluorescence intensity response according to the excitation intensity. In Fig. 2b and c, the logarithm plot clearly showed a slope at around 1 for both ECFP and mTFP (GFP is not tested because its fluorescence signal easily saturates). This result is a strong evidence that both ECFP and mTFP are excited in a single photon excitation manner, even though their emission peaks are at the wavelength region shorter than the excitation laser light. It is especially worth mentioning that a cw laser can be used for the anti-Stokes fluorescence excitation of FPs, thus the same principle can be readily adaptable to any existing fluorescence microscopes to increase the number of imaging channels26.

Hybrid fluorescence-Raman imaging of cells

After demonstrating the feasibility of anti-Stokes fluorescence excitation in purified FP systems, next is to apply the method for the hybrid fluorescence-Raman imaging of HeLa cells that express different FP fusion constructs. Cross-talk free anti-Stokes fluorescence image and cytochrome c, protein and lipid Raman image HeLa cells were successfully obtained from HeLa cells transfected with histone-ECFP (Fig. 3a∼e) and B4GalT1-mTFP (Fig. 3f∼j). Histone is the protein that help pack the DNA inside cell nucleus30. B4GalT1 encodes the β-1,4-galactosyltransferase 1 that helps the synthesis of poly-N-acetyllactosamine, and localise in the trans-Golgi network31. The fluorescence contrast in Fig. 3a and f corresponded well to those of histone and B4GalT1, i.e. cell nucleus and Golgi body respectively. The presented spectra have been processed by SVD denoising, and the anti-Stokes fluorescence images were composed by using the denoised hyperspectral dataset. The different colour spectra presented in Fig. 3e and j were selected from a representative pixel within the corresponding colour images (Fig. 3a∼d and f∼i) of the hyperspectral datasets. To verify whether the anti-Stokes excitation of FPs bring unexpected effects to the fluorescence image, we have also compared the two anti-Stokes fluorescence image with the standard wide-field Stokes fluorescence image of the specimen. The result proved that although the fluorescing mechanism is different, both FP imaging methods give almost identical imaging contrast (Fig. 4). However, slight differences in FP localisation can be observed between the two B4GalT1-mTFP images (Fig. 4f). This is because the standard fluorescence image was taken before the hybrid fluorescence-Raman image, and the movement of Golgi bodies within the short time was recorded. This result also highlights the importance of hybrid imaging. Since living cells are dynamic samples that constantly change their morphology and chemical state, if any time lag exists between the fluorescence and Raman measurements, the accuracy of further chemical analyses according to FP expression pattern would be significantly lowered. Our hybrid imaging technique ensures that the fluorescence and Raman data are fully synchronised, and eliminate such uncertainties.

Figure 3

(a∼e) are the anti-Stokes fluorescence image (a), resonance enhanced 750 cm−1 cytochrome c Raman image (b), 1680 cm−1 amide I Raman image that shows protein contrast (c), 2852 cm−1 CH2 stretch Raman image that shows long chain lipids (d), and the hybrid spectrum taken from a random pixel of the corresponding contrasts (e). Cyan is the spectrum with strong CFP emission, green is with strong cytochrome c contrast, magenta from strong protein contrast and red from strong lipid contrast. Note that all these information are acquired within a single scan of the sample. (f∼j) are the respective images and spectrum of B4GalT1-mTFP labelled HeLa cells as described earlier. All 3 repeats of successfully transfected histone-ECFP HeLa cells and all 6 repeats of successfully transfected B4GalT1-mTFP HeLa cells showed successful combination of fluorescence and Raman imaging as shown here. Also, please note that the presented spectra are without fluorescence background subtraction, but the Raman images are with background subtraction calculations. The width of all images are 40 µm.

Figure 4

(a,b) are the respective anti-Stokes fluorescence image and wide field normal fluorescence image of histone-ECFP labelled HeLa cells, and (c) is the colour merged image of (a), red channel, and (b), green channel. (d–f) are the respective anti-Stokes fluorescence image, wide field normal Stokes fluorescence image and colour merged image of B4GalT1-mTFP labelled HeLa cells. (d) is the red channel and (e) is the green channel in (f). (a,d) shows much lower background signal than (b,e) because the anti-Stokes images are taken in a slit-scan manner, which uses a slit to reject out-of-focus background signals, while (b,e) are wide field images without any restrictions in the detection path. It is worth noting that some of the Golgi bodies are displaced between (d,e), as clearly shown by the white arrows in (f). (f) is merged by optimising the contrast overlap in the top left and bottom right cell. The width of all images are 40 µm.

Another concern regarding the anti-Stokes excitation of FPs in live-cell imaging is how the Stokes emission tail of the anti-Stokes fluorescence emission (Fig. 2) would influence the Raman spectra in the hybrid fluorescence-Raman hyperspectral dataset. To address this issue, first, we compare the spectra taken at the subcellular region with and without anti-Stokes fluorescence signals (Fig. 3e and j, spectra in cyan vs. the spectra with other colours). In histone-ECFP transfected cells (Fig. 3e), the spectrum in cyan showed a very weak anti-Stokes fluorescence peak that is barely stronger than the other spectra at the anti-Stokes side. At the Stokes side, the overall background for the spectrum in cyan is almost identical to the cytosol spectrum (in magenta) across 500~3000 cm−1, indicating that Stokes ECFP emission tail has negligible effect to the Raman spectra. In the case of B4GalT1-mTFP transfected cells (Fig. 3j), the spectrum in cyan shows a much stronger anti-Stokes fluorescence emission than ECFP in Fig. 3e. Due to the strong anti-Stokes fluorescence intensity, even at the Stokes side, the spectrum in cyan has a significantly higher background than the cytosol spectrum (in magenta), which is around the level of the mitochondria spectrum (in green). This indicates that for ECFP and mTFP, the influence of their Stokes emission tail to the cell Raman spectra is at most around the level of autofluorescence background fluctuation in the cells, which can be easily eliminated by background subtraction algorithms. To more convincingly demonstrate the small effect of the Stokes emission tail of the FPs, we compared the hyperspectral images constructed by the anti-Stokes fluorescence emission band, cytochrome c Raman band and fluorescence background at the silent region of the Raman spectra (Sup. 2). The completely distinct imaging contrast between the anti-Stokes fluorescence contrast and Stokes fluorescence background contrast clearly demonstrate that the main contributor for the two fluorescence signals come from different origins. The high similarity of the Stokes fluorescence background contrast to mitochondria, imaged by the cytochrome c contrast, further suggests that the main contributor of the Stokes fluorescence background is indeed autofluorescence32.

To minimise the background influence on the Raman images, the Stokes region of the hyperspectral dataset were extracted and an automated autofluorescence background subtraction method33 was applied before the Raman images were reconstructed (Fig. 3b∼d and g∼i). The cytochrome c, protein and lipid Raman images in Fig. 3 all correspond well to our previously reported Raman images of the corresponding vibrational modes34,35, with minimal cross-talk from the anti-Stokes fluorescence image of the same hyperspectral dataset. This indicates that our hybrid fluorescence-Raman imaging technique indeed provides authentic fluorescence and Raman information for further analysis. The proper combination of FPs and excitation laser is the key of this technique. For example, even under the 532 nm anti-Stokes excitation scheme, the fluorescence signal from GFP is so strong that it overwhelms the Stokes region of the spectra and severe fluorescence signal contamination can be seen in the Raman images (Sup. 3). However, as demonstrated in Sup. 1, by using a different excitation wavelength, it is still possible to use any FP of interest for hybrid imaging. Beside FPs, we have also verified that our method can be applied to chemical probes such as CellTracker (Sup. 4). The hybrid fluorescence-Raman imaging technique is therefore a universal technique that can be applied to most fluorescence imaging studies of living cells to provide the general chemical state of the cell.

The chemical analysis of stem cells according to Oct-4 expression

Being able to image fluorescence and Raman contrasts at the same time means that the setup enables us to correlate the chemical profile of the cell to its protein expression pattern for the first time. Since our previous studies have successfully visualised the chemical evolution of stem cells throughout their differentiation process4,36, here we use stem cell differentiation again as the model system to verify whether our technique is sensitive enough to detect the chemical difference between stem cells across subtle differentiation steps. Transcription factor Oct-4 play a key role in maintaining stem cell pluripotency37. Mouse embryonic stem (ES) cells cultured in medium with leukaemia inhibitory factor (LIF) maintains pluripotency and show high expression level of Oct-4. Once LIF is removed from culture medium, ES cells begin to differentiate with reduced Oct-4 expression37. Here, we established a reporter gene system that uses Oct-4 promoter to drive TFP expression. For ES cells containing the reporter, TFP fluorescence emission would be observed before differentiation, and disappear after they lose their pluripotency38.

The hybrid Raman-fluorescence imaging of ES cells containing the reporter was conducted after LIF was removed from the culture medium for 3 days to let the cells enter their early stage of differentiation. The hyperspectral hybrid fluorescence-Raman datasets of the ES cells again showed negligible cross-talk between the fluorescence and Raman images (Fig. 5a∼e). However, this time a clear photobleach effect of the mTFP signal can be seen (Fig. 5a). Considering the really low absorption cross-section of mTFP under this off-peak illumination condition, such obvious photobleaching phenomenon was unexpected. The reason behind the unexpected photobleaching rate might be due to the overlap of the incident laser to both the absorption and emission spectra, which resulted in the stimulated cycling between the fluorophore excited state and ground state. It is possible to reduce the laser intensity to minimise such effect (Sup. 5), but the signal-to-noise ratio of the Raman spectra will also decrease, thus reduce the quality of the chemical information presented in the Raman spectra. Comparing the anti-Stokes fluorescence image and transmission optical image of the ES cells (Fig. 5a and f), we show that the ES cells have come to the differentiation stage that even within the same colony, part of the cells has already lost their pluripotency (hence the inactivation of Oct-4) and the other part of the cells are still in the course of differentiating (hence the expression of mTFP through an active Oct-4 promoter).

Figure 5

(a∼e) are the anti-Stokes fluorescence image (a), resonance enhanced 750 cm−1 cytochrome c Raman image (b), 1680 cm−1 amide I Raman image that shows protein contrast (c), 2852 cm−1 CH2 stretch Raman image that shows long chain lipids (d), and the hybrid spectrum taken from a random pixel that strong anti-Stokes fluorescence signal can be seen in the field of view of mouse ES cells transfected with pOct4-mTFP. (f) shows the wide field optical image of the ES cells. By comparing (a,f), it is clear that some cells in the colony express Oct-4 while the others do not. (g,h) shows the DAPC result for ES cells expressing (Oct-4+) and not expressing (Oct-4−) Oct-4 using linear (g) and quadratic (h) discrimination algorithms. Each spot represents the averaged spectrum from one cell. DAPC is able to roughly discriminate ES cells with different Oct-4 expression pattern with a 25.71% and 16.67% error rate in (g) and a 8.57% and 8.33% error rate in (h). (i) shows the F1 vector that represents the most dominant spectral difference between the Oct-4+ and Oct-4− groups. The width of all images are 40 μm.

Since our new hybrid setup can simultaneously acquire the Raman spectra of these ES cells, we continued to verify whether the Stokes Raman spectra is sensitive enough to identify the subtle chemical difference between these ES cells with different expression pattern of Oct-4, but at close differentiation steps. The spectral analysis method we choose for the purpose is the discriminant analysis of principle components (DAPC)39,40. The linear discrimination plot showed a 25.71% error rate to correctly categorise an Oct-4 expressing (Oct-4+) cell, and a 16.67% error rate to correctly categorise an Oct-4 non-expressing (Oct-4−) cell (Fig. 5g); while the quadratic discrimination plot showed a 8.57% error rate to correctly categorise an Oct-4+ cell, and a 8.33% error rate to correctly categorise an Oct-4− cell (Fig. 5h). To further confirm the statistical significance of the result, we applied Wilks’ lambda test to our result and got a p-value of 0.0087%. This demonstrates that our method can indeed guide Raman spectroscopy to discriminate the tiny chemical shift related to the on-and-off of a specific gene, in this case Oct-4. Furthermore, during DAPC calculation, an F1-vector is drawn between the centres of the two groups in the PC hyper-space. This F1-vector is considered to represent the largest spectral difference in between the two groups after all the meaningful PCs have been taken into account (Fig. 5i). The F1-vector is basically fluorescence background free, suggesting that the fluorescence emission tail at the Stokes region is not the key factor that separates the 2 groups. However, considering that some biomolecules may be more abundant in Oct-4+ cells and others more abundant in Oct-4− cells, the spectral profile of the F1 vector would be a mix of positive and negative Raman spectral components from these differences, making the F1 vector difficult to interpret. More chemical details might be revealed with the further advancements in multivariate spectral analysis techniques to help resolve the detailed spectral components in the F1 vector.

Chemical analysis of the fluorescence labelled sub-cellular compartments

Besides the chemical profiling of living cells according to protein expression pattern, another important application of the hybrid imaging technique is to use FPs as a guide for the chemical analysis of living cells. Here, we use the hybrid fluorescence-Raman images of histone and B4GalT1 labelled HeLa cells in Fig. 3 as example. First, about the histone-labelled HeLa cell in Fig. 3a, the averaged Raman spectra within and outside of the labelled cell nuclei in Fig. 3a can be easily extracted (Fig. 6a). For the averaged Raman spectra of the non-fluorescing compartment, the pure culture medium spectra that does not contain any cellular information are excluded to ensure we are comparing the spectral content within the cellular region. It is not difficult to see that even though cell nuclei are considered to have a significant difference in its nucleic acid content compared with cytosol, the general morphology of the nucleus spectrum is still somehow similar to the averaged spectrum of the out-of-nucleus region (Fig. 6a). The detailed chemical differences can be more easily identified by calculating the difference spectrum. By subtracting the nucleus spectrum with the out-of-nucleus spectrum (Fig. 6b), we can easily identify that the cytochrome c Raman bands (750 cm−1, 1132 cm−1, 1587 cm−1) and lipid Raman bands (1266 cm−1, 1302 cm−1, the sharp 1656 cm−1 peak, 2852 cm−1) point to the negative direction, while the protein Raman bands (1004 cm−1, the broad 1656 cm−1 band, 2950 cm−1) and nucleic acid Raman band (780 cm−1) points towards the positive direction. The result corresponds to our general understanding that cell nucleus is rich in protein and nucleic acid content, while having less cytochrome c and lipid content than the other cellular compartments.

Figure 6

(a) Compares the averaged Raman spectra in the histone-ECFP expressing region and the other cellular area in Fig. 3a. The histone-ECFP expressing region is basically equivalent to cell nuclei. Their spectral difference is shown in (b). The * mark labels the negative cytochrome c peaks, while L, P, N labels the lipid, protein and nucleic acid Raman peaks, respectively. (c) compares the averaged Raman spectra in B4GalT1-mTFP expressing region and the other cellular area in Fig. 3f. The B4GalT1-mTFP expressing region is basically equivalent to Golgi body. Their spectral difference is shown in (d). The Ls label the lipid Raman peaks that could also be identified in (b). (e) Shows the Raman spectrum of a lipid droplet in the hyperspectral dataset of Fig. 3f∼j. All spectral features seen in (d) can be seen in (e), causing the spectral difference difficult to extract by multivariate analysis means.

Next, we used the same method to analyse the chemical content of Golgi body in living cells. It is worth mentioning that no unsupervised multivariate spectral analysis is able to discriminate Golgi body from its surroundings yet. The averaged Raman spectrum of the fluorescing compartment in B4GalT1-mTFP expressing cells (Fig. 3f) and the non-fluorescing area were calculated (Fig. 6c). Again, the two spectra look highly similar to each other at the first glance. To visualise the subtle chemical difference, the difference spectrum between the Golgi body and the other compartments is calculated (Fig. 6d). The broad background in the difference spectrum most likely originates from the mTFP fluorescence tail in the Stokes region, and the wave-like structure below 1100 cm−1 is most likely due to the spectral profile of the notch filter. Such wave-like spectral pattern does not appear when the spectral difference is below 15 CCD counts (Sup. 6). Above 1100 cm−1, the spectral profile clearly shows the spectrum of lipids. The 1266 cm−1, 1302 cm−1, 1656 cm−1, and 2852 cm−1 Raman bands can all be seen in the negative spectral component in the nuclei difference spectra as well (Fig. 6b). Together with the 1440 cm−1 Raman band, these can all be assigned to lipid bands. Since the spectral difference is similar to the Raman spectrum of lipid droplets (Fig. 6e), but at a much lower contrast, it is very difficult to extract the information by multivariate analysis means. Repeat experiments show consistent lipid-like difference spectrum for Golgi body analysis, while many of them also exhibit the Raman spectral features of cytochrome c (Sup. 6). This indicates that Golgi body might also have a tendency to attach to mitochondria in HeLa cells.

— Nature Scientific Reports