update on Nikon objective immersion oils

August 30, 2018 at 8:41 am | | everyday science, hardware, review

A few years ago, I compared different immersion oils. I concluded that Nikon A was the best for routine fluorescence because: (A) it had low autofluorescence, (B) it didn’t smell, (C) it was low viscosity, and (D) the small plastic dropper bottles allowed for easy and clean application.

Unfortunately, my two favorites, Nikon A and NF, were both discontinued. The oil Nikon replaced these with is called F. But I don’t love this oil for a few reasons. First, it’s fairly stinky. Not offensive, but I still don’t want my microscopes smelling if I can help it. Second, I’ve heard complaints from others that Nikon F can have microbubbles (or maybe crystals?) in the oil, making image quality worse. Finally, dried F oil hardens over time, and can form a lacquer unless it is cleaned off surfaces very well. That said, F does have very low fluorescence, so that’s a good thing.

I explored some alternatives. Cargille LDF has the same optical properties as Nikon F (index of refraction = 1.518 and Abbe Ve = 41). But LDF smells terrible. I refuse to have my microscope room smell like that! Cargille HF doesn’t smell and has similar optical properties, but HF is autofluorescent at 488 and 405 nm excitation, so it adds significant background and isn’t usable for sensitive imaging.

At the recommendation of Kari in the UCSF microscopy core (and Caroline Mrejen at Olympus), I tried Olympus Type F, which also has an index of refraction of 1.518 and an Abbe number of 40.8, which is compatible with Nikon. The Olympus oil had very low autofluorescence, on par with Nikon A, NF, and F. (I also tested low-fluorescence oils Leica F and Zeiss 518F, but their dispersion numbers are higher (Ve = 45-46), which can cause chromatic aberration and may interfere with Perfect Focus.)

I used to love the low viscosity of Nikon A (150 cSt), because it allowed faster settling after the stage moved and was less likely to cause Perfect Focus cycling due to mechanical coupling to thin or light samples, plus it was easier to apply and clean. Nikon NF was higher viscosity (800 cSt). Olympus F is higher than Nikon A (450 cSt), but acceptable.

Finally, Olympus F comes is an easy to use applicator bottle: instead of a glass rod that can drip down the side of the vial if you’re not careful, the Olympus F is in a plastic bottle with a dropper. It’s not quite as nice as the 8 cc dropper bottles that Nikon A used to come in, and I don’t love the capping mechanism on the Olympus F, but I’ll survive.

I plan to finish up our last bottle of Nikon A, then switch over to Olympus F. We also have a couple bottles of Nikon NF remaining, which I will save for 37C work (the higher viscosity is useful at higher temperatures).

Belkin Conserve Switch is great for scopes

February 23, 2018 at 10:47 am | | hardware, review

I thought I’d pass along one of my favorite tips: I have several of these Belkin Conserve Switch power strips in lab. I use them to turn on a scope and all its peripherals with a flip of one single switch!

You can set one switch to power multiple strips. It’s so much better than flipping on each piece of equipment (and inevitably forgetting one)! You just need to check with the equipment manufacturer if it’s safe to power on/off the item by basically unplugging it.

2017 nobel prize predictions

September 20, 2017 at 10:14 am | | nobel

It’s approaching Nobel season again, and here are my predictions:

Chemistry: CRISPR (Doudna, Charpentier, Zhang)

Medicine: Unfolded protein response (Walter, Mori)

Physics: Gravitational waves (Kip Thorne, Rainer Weiss, Ronald Drever, or maybe Barry Barish and the entire LIGO collaboration)

Last year, I think the detection gravitational waves happened a little too late to actually be selected for 2016. But now it’s a year later! Unfortunately, Ronald Drever passed away in the meantime.

In years past, I think CRISPR’s potential had not been actualized enough to win, but by this time it’s obvious that the technology works and is already impacting science. Lithium batteries have changed the world, and John Goodenough deserves the prize. But he recently announced a new battery technology that some scientists are skeptical will work. Maybe that’s too much controversy for the Nobel committee?

I considered optogenetics (Deisseroth, Zemelman, Miesenböck, Isacoff), but I didn’t want to predict both that and CRISPR in one year. Since Peter Walter and Kazutoshi Mori won a Lasker prize a few years ago now, I think it’s their time.


My past predictions

Clarivate (formerly Thompson) Citation Laureates

C&E News webinar


Stat News

As always, excellent prediction and discussion at Curious Wavefunction

Photometrics Prime95B demo number 2

September 13, 2017 at 2:49 pm | | hardware, review

Technical Instruments loaned me a Photometrics Prime95B back-thinned CMOS camera. I had demoed this camera before, but I was able to put it on our scope this time. Our spinning disk confocal has two camera ports, so I installed a tube lens that made the effective pixel size on the Prime95B approximately the same as our 512×512 Andor iXon EMCCD. The Prime95B looked beautiful for a moderately bright sample:

(Note that I cropped the Prime95B images by approximately 60% both laterally and axially, because the illumination area on the microscope was restricted to the center of the field of view. Uncropped, the Prime95B field of view would be over twice as big in each dimension!)

At very low light imaging, I had to set the EMCCD gain very high to get an image with good signal-to-noise. The Prime95B had slightly lower sensitivity in this imaging regime, but honestly, I was surprised that its images looked that good:

The only problems I ran into had to do with the PVCAM driver for the camera having some issues in Micro-Manager (mainly with having trouble shuttering the lasers correctly), but I was able to find moderately acceptable workarounds.

If I were buying a camera for spinning disk, TIRF, epifluorescence, etc. (really, anything except single-fluorophore microscopy), I would probably get a Prime95B. I hope other sensor manufacturers and scientific camera companies follow suit and release more excellent back-thinned CMOS cameras.

review of Point Grey camera for microscopy

February 28, 2017 at 5:40 pm | | hardware, review

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.

Also, Kurt tested a different Point Grey camera with great results.


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.

always have plastic sheeting in lab

October 24, 2016 at 10:46 am | | everyday science, hardware, stupid technology

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. :(

2016-09-02 14.27.09

2016-09-02 14.17.25

For less than $5, you can get some rolls of the stuff. If you want larger and thicker sheets (like in the photos), I recommend this stuff.

2016 nobel prize predictions

September 9, 2016 at 11:39 am | | nobel

UPDATE: Turns out the Simpsons were right once again

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.)

Other predictions:

Thompson ISI

Curious Wavefunction

In the Pipeline

Transcription and Translation

electrically tunable lenses for microscopy

September 2, 2016 at 2:22 pm | | hardware, literature

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.


etl z stack

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.

etl light sheet

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.

etl light sheet 2

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.

  1. 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

  2. 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

update on scope room dustiness

August 26, 2016 at 4:39 pm | | hardware

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.)

air 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.

UPDATE (1/17/17):

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:

demoing the Photometrics Prime 95B back-thinned CMOS

August 1, 2016 at 4:10 pm | | hardware, review

(See my second post on this camera here.)

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.

emccd vs prime bead

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.

emccd prime

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!

review of NanoLive microscope

July 12, 2016 at 1:54 pm | | hardware, review

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)
  • label-free
  • 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!

experimenting with preprints

May 9, 2016 at 12:15 pm | | literature, science community

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.

2015 nobel in medicine: parasites

October 5, 2015 at 4:28 pm | | nobel

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.

2015 nobel prize predictions

September 9, 2015 at 1:48 pm | | nobel

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

home-built coverslip drier/spinner

April 3, 2015 at 4:14 pm | | hardware

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):

2015-04-03 11.42.00

And here’s the finished product.

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