I bought a $500 camera from Point Grey that has the Sony IMX249 chip. It is a fairly large field of view with intermediate sized pixels (5.86 um), so it has a great dynamic range. The great thing is that it has low dark/read noise of 7-14 electrons per frame and a very high quantum efficiency of 80%. At it runs at up to 40 fps!
While this camera can’t fully compete with scientific CMOS cameras like the Andor Zyla or Hamamatsu Flash4 (and definitely not with the Photometrics Prime95B), because these scientific cameras do a better job cooling (reducing dark counts) and on-chip correction of dead pixels or other pixel-to-pixel variability. But I wondered if this Point Grey camera could be a very cheap replacement for our old interline CCD (a Hamamatsu Orca-ER model C4742-80-12AG).
Recently, Nico wrote a Micro-Manager device adapter for USB3 Point Grey cameras, so I quickly bought the Blackfly BFLY-U3-23S6M-C and was happy to get beautiful images! The picture on the bottom is from the Point Grey and the one on the top is from the old interline camera. At the same exposure time, the images were very similar. And the Point Grey camera could run 4x faster if necessary.
In addition, the Point Grey outputs 16-bit-images with much higher dynamic range than the old 12-bit interline CCD. I misread the specs: the video output is 16 bit, but the A/D converter is still only 12 bit.
So I plan to replace the interline camera with this Point Grey camera for day-to-day microscopy. I’ll let you know if we run into any problems in the future.
Here are two more images, zoomed in and cropped. Top is Hamamatsu Orca-ER and bottom is the Point Grey Blackfly camera:
These were the same exposure time (200 ms) and the same magnification. I’ve decided to replace the Hamamatsu with the Point Grey camera.
I learned during my PhD that you should always have plastic sheeting in lab, because it might just save your equipment when/if a water leak happens. It saved one of our scopes recently, although I wasn’t fast enough to prevent some water damage on an expensive camera. :(
Time for my 2016 Nobel Prize predictions:
Chemistry: Lithium-ion batteries (John Goodenough, Stanley Whittingham, Akira Yoshino)
Medicine: T-cell receptor (James Allison, Ellis Reinherz, Philippa Marrack)
Physics: Gravitational waves (Kip Thorne, Rainer Weiss, Ronald Drever, or maybe the LIGO collaboration)
Last year, I played the CRISPR card and lost. Also, I guess that I must stop saying “Ron Vale for kinesin” over and over again. So I tried to keep things fresh this year, but both my medicine and chemistry predictions are repeats.
For physics, I’d like to see the prize go to the entire LIGO collaboration, considering that there were thousands of scientist involved in demonstrating Einstein’s predictions. But I understand why the Nobel committee would prefer to award it to individuals, and there are 3 who are kinda obvious. 2016 might be too early for this award, considering the nominations are due Feb 1, but probably someone knew the gravitational waves discovery was imminent and nominated them? Or maybe my prediction is wrong, and it will exoplanets this year.
For chemistry, I think polymer synthesis could win, but it might not be sexy enough. I think batteries have demonstrated their impact on the world of portable electronics and electric cars. And Goodenough is old. I know I’ve predicted batteries in the past, but I hope I’m right this time!
Hopefully Nature doesn’t make fun of me again this year.
(See my past predictions and discussions here.)
Electrically tunable lenses (ETLs) are polymeric or fluid-filled lenses that have a focal length that changes with an applied current. They have shown some great potential for microscopy, especially in fast, simple z-sweeps.
The above figure shows the ~120 um range of focal depths an ETL installed between the camera and a 40x objective (from reference 1). Note that this arrangement has the drawback of changing the effective magnification at different focal depths; however, this effect is fairly small (20%) and linear over the full range. For high-resolution z-stack imaging of cells, this mag change would not be ideal. But it should be correctable for imaging less sensitive to magnification changes. Basic ETLs cost only a few hundred dollars, a lot cheaper than a piezo stage or objective focuser. Optotune has a lot of information about how to add an ETL to a microscope.
Another cool application of an ETL is in light-sheet microscopy. A recent paper from Enrico Gratton (reference 2) used an ETL to sweep the narrow waist of a light sheet across the sample, and synchronize its motion to match the rolling shutter of a CMOS camera.
The main goal was to cheaply and simply create a light sheet that had a uniform (and minimal) thickness across the entire field of view. Previous low-tech methods to achieve this was to close down an iris, thus reducing the difference in thickness across the sample, but it also reduces the minimal waist size. The high-tech way to do this is creating “propagation-invariant” Bessel or Airy beams. These do not spread out as they propagate, like Gaussian beams do, but creating them and aligning them in microscopes is significantly more challenging.
Gratton’s cheap trick means one can create a flat and thin light sheet for the cost of an ETL and the complexity of synchronizing a voltage ramp signal to the CMOS rolling shutter readout. To be honest, I don’t 100% know how complicated or robust that is in practice. I’m just guessing that it’s simpler than a Bessel beam.
Wang, Z., Lei, M., Yao, B., Cai, Y., Liang, Y., Yang, Y., … Xiong, D. (2015). Compact multi-band fluorescent microscope with an electrically tunable lens for autofocusing. Biomedical Optics Express, 6(11), 4353. doi:10.1364/BOE.6.004353
Hedde, P. N., & Gratton, E. (2016). Selective plane illumination microscopy with a light sheet of uniform thickness formed by an electrically tunable lens. Microscopy Research and Technique, 00(April). doi:10.1002/jemt.22707
A while ago, I installed a very simple filter for the air vent in our scope room. It barely did anything, honestly. The “filter” is nothing more than a very loose mesh of fibers.
So I’ve bumped up to a 20x20x1 inch MERV13 pleated air filter with a paperboard frame. It fits perfectly between the vent and the grate. I even did some actual tape-measuring before purchasing. :) With a little duct tape, I was able to secure and seal the filter into place, then I put the grate back on. (A 16x16x1 inch filter would have fit, too, but I would have had to remove the louvers on the vent before taping on the filter.)
I didn’t notice any reduction of air flow and the room is still under positive pressure. So I’m not concerned with straining the HVAC system of the building.
I hope that it will help keep down the dust in the scope room! I’ll check it in a few months and update.
I’ve changed the filter twice since August. Here’s a side-by-side of the new filter and filter that’s been installed for a few months. I think it’s working:
Photometrics has released the Prime 95B, the first scientific CMOS camera with a back-thinned sensor. This means that the sensor is significantly more sensitive than the front-illuminated versions of other CMOS scientific cameras. So the Prime 95B has a 95% quantum efficiency, whereas other scientific CMOS cameras have 60-70% QE; the newest version of competing CMOS cameras tout 80%+ QE. Back-thinning really helped CCD technology (EMCCDs are back-thinned, for example), but back-thinning CMOS sensors has been more challenging, for some technical reasons that I don’t know.
I demoed the Prime 95B when it was in the Nikon Imaging Center (Kurt wrote up details here). The CMOS camera was installed on a spinning disk confocal along with a 1024×1024 pixel EMCCD. The Prime 95B has 11 um pixels, slightly smaller than the 13 um of the EMCCD’s pixels; this results in a higher spatial sampling rate and thus lower sensitivity for the CMOS, because the photons are spread across more pixels. This can be simply corrected by using a different lens, but we didn’t do that here. So it provided an unlevel playing field, favoring the EMCCD.
Despite that, the Prime 95B matched or outperformed the EMCCD in all the tests we did. The above image compares the EMCCD (left) with the Prime 95B (right) imaging a 100 nm Tetraspeck bead. Below, I compare them imaging a fixed test sample at very low light levels.
The comparisons I made were mainly qualitative. By eye, I was not able to find conditions were the EMCCD outperformed the Prime 95B. That’s saying a lot, especially because the Prime 95B costs approximately half as much! For single-molecule imaging, the EMCCD might still be the king (see Kurt’s curve), but I didn’t have time to perform those detailed or quantitative tests. But for all other imaging and spinning disk confocal, I’d rather have the Prime 95B. No more deciding the optimal EM gain settings and the large dynamic range of the CMOS make it a real winner!
We got a chance to try out a cool new label-free microscope from NanoLive: the 3D Cell Explorer. It works on a holographic tomography, by rotating a laser beam around the top of the sample and records many transmitted-light images. It then uses software to reconstruct the image with phase and even 3D information. The small index differences of different organelles or regions of the cell results in different retardation of the phase of the transmitted light; in the reconstruction, these areas can be false-colored to give beautiful renderings of cells … all without fluorescent labeling.
We used the Nanolive to watch Naegleria amoeba crawling across a glass surface. These cells move orders of magnitude faster than fibroblasts (20 um/min), so imaging their movement is a serious challenge for many high-resolution microscopes.
The above video is false-colored for different index ranges. It is super cool to see the pseudopods in 3D, and possibly even distinguish the plasma membrane from the actin cortex. The demo went well and it took only about 15 min to take the microscope out of the box and start imaging.
When we demoed the beta version a year or so ago, and it had trouble imaging crawling amoebae: the background subtraction was manual and flaky and the frame rate was too slow. But Nanolive let us try it again after the actual release of the product and things works way better. The background subtraction is now automated and robust, and the frame rate was high enough to watch these fast crawling cells.
I think that this microscope would be a great choice for researchers studying organisms that are not genetically tractable or otherwise cannot be fluorescently labeled. Or for anyone studying organelles that show up with a different index (Naegleria ended up having relatively low-contrast organelles compared to adherent mammalian cells, for instance.)
- affordable (about the cost of an EMCCD camera)
- low intensity (no phototoxicity or photobleaching)
- simple and user-friendly: easier that setting up DIC in Koehler illumination :)
- small footprint and easy setup
- software is free
- potential for beautiful and amazing data
- not versatile: it does one thing (but does that one thing well)
- limited to samples with wide top, like a 35 mm dish (not 96-well plates), because the laser beam comes in at an angle
- 3D information on top and bottom of cells is less impressive
Go check it out!
We recently uploaded a preprint to bioRxiv. The goal was to hopefully get some constructive feedback to improve the manuscript. So far, it got some tweets and even an email from a journal editor, but no comments or constructive feedback.
I notice that very few preprints on bioRxiv have any comments at all. Of course, scientists may be emailing each other privately about papers on bioRxiv, and that would be great. But I think a open process would be valuable. F1000Research, for example, has a totally open review process, posting the referee reports right with the article. I might be interested in trying that journal someday.
I love this year’s Nobel Prize in Medicine. Curious Wavefunction has a great writeup on it. I love that it was awarded for efforts to help prevent painful and fatal diseases the inflict millions around the world, especially in poorer countries. I also love that it recognizes how Artemisinin was derived from traditional medicine, but then isolated and tested for effectiveness and safety. There needs to be more of that. Traditional medicines may certainly be effective, probably because they contain some active drug that influences the body. But I don’t want to take unknown doses of unknown chemicals please.
Time for Nobel Prize predictions. (See my past predictions and discussions here.) My 2015 predictions:
Chemistry: CRISPR: Doudna, Charpentier
Medicine: Immune Cancer Therapy: James Allison, Michel Sadelain
Physics: Electromagnetically Induced Transparency: Lene Hau, Steve Harris
Peace: Ebola: Médecins Sans Frontières
We wanted some coverslip spinners to dry coverslips after washing and rinsing. It’s way faster than blowing them with air. Nico kindly gave me his 3D design file for the coverslip holder, and I modified the box design from here.
Here’s a parts list (Digikey part numbers unless otherwise noted):
- Power supply/AC adaptor (
3V, 500mA5V, 1A)
- 3V DC motors (2)
- wired in parallel
- Momentary switch
- Cushion feet (4)
- Screws for motors
- Mcmaster-Carr part 90116A009
- 3D print coverslip holders (2)
- Safety cover
- Superglue, wire, soldering iron, etc.
And here’s the finished product.
Most importantly, I thank my advisor, W.E. Moerner. It is difficult to explain how wonderful it has been to study under him. W.E. is a real scientist’s scientist: he fundamentally cares about good science and presenting results in a clear and honest manner. He always impressed me with his understanding of sciences outside his field and his scholarship, as I doubt that there is any paper I have read that he has not. W.E. always knows where some obscure piece of equipment is in the lab, and what type of power cable it requires. W.E.’s humor and generosity have been invaluable during my time in his lab, not to mention his scientific guidance. I could not have asked for a better Ph.D. advisor.
I joined the Moerner lab because W.E. seemed to run a fun and exciting research program, and I have not been disappointed. Other members of the Moerner Lab have been instrumental in my education and research. Kallie Willets mentored me when I first arrived at Stanford. Kallie was fun to work with and I am very grateful for the time and energy she dedicated to helping me get a solid footing in the lab by teaching me the right way to do things (and clean up afterwards).
After Kallie graduated, it was entertaining (to say the least) to get to know my officemate Dave Fromm. Dave was always willing to discuss problems I was facing in my experiments, and often suggested perfect solutions. (He was also always willing to discuss his adventures and funny stuff he found on the internet.) Dave and Jim Schuck regularly played darts over my head … literally. In general, this was entertaining and helpful to my overall spirit, and I appreciate the fun times with Jim and Dave. In those early years, I also enjoyed the company of (and scientific input from) Nick Conley, Anika Kinkhabwala, Adam Cohen, Stefanie Nishimura, Jaesuk Hwang, Kit Werley, So Yeon Kim, Andrea Kurtz, Marcelle Koenig, and Jian Cui.
In the later years of my tenure in the Moerner Lab, I have benefited from another batch of amazing people. Nick is one of the most motivating collaborators I have had the pleasure of working with; he is always excited about results, and his mind wanders to great places (not to mention that his skills as an organic chemist were very helpful to me)! I also had the opportunity to work with Hsiao-lu Lee, who was always generous with her time and expertise in cell culture. I am thankful to have those two wonderful coauthors. Alex Fürstenberg has been a fun (and very tolerant) officemate, and is always a great person to ask about anything photophysical. Mike Thompson is hard working and smart, but most importantly he laughs at more than 83% of my jokes. Julie Biteen is opinionated and usually right, and has been fun to bounce ideas off. All the other members of the Moerner Lab (Shigeki, Randy, Majid, Steve, Jianwei, Whitney, Lana, Yan, Sam B, Quan, Matt, etc.) are exceptional people and have made Stanford a wonderful place.
Marissa Lee started joined the lab in 2008, joining my project. I have enjoyed mentoring her and passing on as much as possible of what Kallie, Dave, Jim, Stefanie, Nick, Hsiao-lu, So Yeon, Jaesuk, Adam, and Anika taught me over the years. I wish her luck in her time at Stanford. Several summer students worked with me to get a taste of research. I thank Jennifer Alyono, Daniel Lau, Nathan Hobbs, and John Servanda for their help taking spectra.
Of course, I must also acknowledge Bob Twieg and his students at Kent State University. As a physical chemist, there is nothing better than an excellent collaboration with a group of top-notch synthetic chemists. W.E. and Bob have worked together since their IBM days in the 1980s and 1990s, and I had the fortune to benefit immensely from that bond between labs. Nearly every compound mentioned in this Dissertation was synthesized by the Twieg lab, and the back-and-forth (or push– pull?) design process between the labs should serve as an example to what all collaborations should strive for. Bob’s students have made great compounds over the years, and I thank all of them for being super collaborators: Meng, Hui, Zhikuan, Na, Reichel, Ryan, Alex, and Jarrod.
Friends have made grad school a blast. I met the Moilanens immediately, and enjoyed marathon training and adventures with David and Hailey. Ben Spry was a great help studying for placement exams, and I enjoyed driving to San Jose with Ben so he could buy a Camaro. William Childs and Charles McCrory—after I finally decided to like them—were indispensible: grad school will be filled with fond memories of coffee, lunch, and arguments because of Wm and Charles. So many other friends made my time at Stanford wonderful: Nichole, Kate, Alicia, Jen, Drew, Ashley, John, Zalatan, Chad, Matt, Griffin, Kendall, Daniel, Adrienne, Adam, Avisek, Eric, Ethan, Kevin, Emily, Ken, Dan, Scott, and everyone else! It has been fun having Jordan and Maria in California, and so many other non-Stanford friends that I cannot possibly name them all. I have had positive interactions with several faculty members, and I thank Bob Waymouth, Chris Chidsey, Dick Zare, Bianxiao Cui, Steve Boxer, Justin DuBois, Vijay Pande, Bob Pecora, and Ed Solomon. I also must recognize the members of the Stanford staff who contributed to my work and enjoyment, namely: Roger Kuhn, Todd Eberspacher, Brian Palermo, Patricia Dwyer, Grace Baysinger, Steve Lynch, and all the Conways—Marc, Daragh, and Mariette.
I feel that I must also acknowledge those in my past who influenced me and led me down the path of science. My earliest memories of enjoying the natural world were at Audubon’s Mast Landing Camp, playing and learning about nature with Aaron and Ira and Matt. In the third grade, Mrs. Solari recognized and encouraged my inclination toward science, as have many teachers since. I thank Dr. Root, who mentored me for my 7th-grade science fair project; Mr. Plummer for dealing with 8th graders; Mr. Glick for the astronomy and recycling clubs and supporting me throughout high school; Mr. Herrick, for being the best physics teacher I never had; Mr. Gauger for insisting that Heisenberg’s uncertainty principle can explain why things still jiggle at zero Kelvin; John Anderson for arguing with me; Don Cass for teaching my first college chemistry class and making it so exciting; Tony Planchart for teaching biochemistry in a way that convinced me to be a chemistry major; Helen Hess for fun classes biology and biomechanics; Michael Rubinstein for his entertaining exploration of polymer physics; Royce Murray for teaching analytical chemistry; Max Berkowitz for stat mech classes; and Charles Schroeder, Eric Shaqfeh, and Steve Chu for a great summer research experience. I should offer a special bit of gratitude to Sergei Sheiko, whose lab I worked in as an undergrad, and who helped make my time at UNC spectacular.
This Dissertation is dedicated to my family: the Lords, the Cyrs, and the Hearns. My parents have always encouraged my interests, without pushing me too hard. I wouldn’t be half the person I am without their support. My brother Jackson has been a life-long companion, so I was very pleased when he moved to California and we could play together like when we were growing up. My grandparents Lord funded my education, which I greatly appreciate. I probably get some of my curiosity from my pépère Cyr. My first year at Stanford, I met Brenna Hearn and married her a few years later. She has made my life wonderful, and I thank her for her support throughout grad school. I cannot thank Brenna enough for her companionship, so I’ll stop there.
Looking back at this, I wish that I had made it 50 times longer and cut out the rest of the dissertation.
LED illumination is awesome for epifluorescence. No mechanical shutters, no changing mercury lamps every 200 hours, no hot lamphouses, no worries about letting it cool down before turning the lamp back on, less wasted electricity, immediately ready to use after turning it on, etc.
We have a Lumencor SpectraX on our Nikon TE2000 scope and we love it. It contains multiple LED that are independently triggerable. For high-speed imaging, we bought one new Chroma quad-band dichroic and emission filter set, as well as 4 separate single-band emission filters for our emission filter wheel (although this latter set is not absolutely necessary).
The amazing thing is to be able to run color sequences at the frame rate of the camera (because the SpectraX accepts TTL triggering of each line independently). It is beautiful to see the rainbow of light flashing out of the scope at 20+ frames per second!
We haven’t run into any issues with brightness: the SpectraX is bright enough for all our cell imaging experiments. Typically, we run it at 20% power. That said, I’m aware that the very bright peaks in an arc lamp spectrum (e.g. UV, 435, 546) aren’t there in the LED spectra. So for FRAP or something, you may not be able to bleach as fast.
And, of course, a fancy illuminator like the Spectra X is not cheap. But for run-of-the-mill epi imaging, white-light sources like the Lumencor Sola might be a good option. Another downside is that the fans on the Spectra X are audible, but not annoying. Despite that minor issue and the cost, I highly recommend LED illumination (and the Spectra X, specifically).
I recommend you demo a few LED sources from a few companies (e.g. ScopeLED, Lumencor, Sutter, etc.) and make sure it will fit your needs.
* Make sure your camera supports TTL triggering of an external shutter.
Jürgen Schmied from GATTAquant came by the other day and let me play around with some of their cool DNA origami fluorescence standards.
The PAINT sample was really cool. It has short oligos on the DNA origami and complementary strands labeled with dyes in solution. The binding/bleaching kinetics are such that each hotspot blinks many times during an acquisition. After a quick 10,000 frame acquisition over 3 min, we collected a dataset that we could easily get a super-resolution image. We used ThunderSTORM to fit the data and correct for drift. But without any other corrections, we could easily resolve the three PAINT hotspots on each DNA origami:
But my favorite sample was actually the confocal test slide. It had two sets of dyes about 350 nm apart permanently labeled on each DNA origami.
This let me test the resolution and image quality using different configurations on our Diskovery confocal/TIRF system.
Each spot contained only about 4-8 dyes. So it was a much greater challenge to our microscope than TetraSpeck beads.
I highly recommend GATTAquant test samples. Very fun.
UPDATE: Jürgen ran my data though GATTAquant’s analysis software and sent me the results below.