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).

UPDATE:

Some people claim that type A was simply renamed type N. I don’t think that’s true. First of all, I couldn’t get Perfect Focus on our Ti2 to work with Nikon type N oil. Second, the autofluorescence of Nikon type N (right) was way higher than Olympus type F (left) or the old Nikon type A, at least at 405 and 488 nm:

So I’ll stick with Olympus type F. :)

UPDATE 2:

Here are some example images. These are excited with 640 (red), 561 (red), 488 (green), and 405 nm (blue) and the display ranges are the same for each sample. (The dots are single fluorophores on the glass.) You can see that Cargille HF is slightly more autofluorescent (especially at 405 nm) than either the old Nikon A or Olympus type F. This matches what Cargille states for HF: “Slightly more fluorescent than Type LDF.”

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.

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.

UPDATE:

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.

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.

nanolive

nanolive2

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.)

Pro:

  • 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

Con:

  • 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!

LED illumination review

March 3, 2015 at 4:06 pm | | hardware, review

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!

https://micro-manager.org/wiki/Hardware-based_synchronization

We use a ESio TTL* box controlled by Micro-Manager and it works great. But you could use an Arduino and some simple wiring using a DE15 breakout board to accomplish the same thing for cheaper.

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.

GATTAquant microscopy standards

February 13, 2015 at 5:27 pm | | review, single molecules

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:

Screen Shot 2015-02-13 at 5.12.44 PM

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.

Screen Shot 2015-02-13 at 5.13.05 PM

This let me test the resolution and image quality using different configurations on our Diskovery confocal/TIRF system.

Screen Shot 2015-02-13 at 5.13.21 PM

Each spot contained only about 4-8 dyes. So it was a much greater challenge to our microscope than TetraSpeck beads.

Screen Shot 2015-02-13 at 5.12.55 PM

I highly recommend GATTAquant test samples. Very fun.

UPDATE: Jürgen ran my data though GATTAquant’s analysis software and sent me the results below.

export3_image

export3_FWHM histogram

export3_Distance histogram

plasma cleaner review

December 18, 2014 at 6:09 pm | | hardware, review

I had previously made my own plasma cleaner using a pump and an old microwave. While my homemade version technically worked, it was complicated to use, unwieldily, and inconsistent in performance. In fact, at least one test made my glass coverslips dirtier.

So we purchased a Harrick plasma cleaner. I’ve used these in the past for preparing coverslips for single-molecule imaging as well treating coverslips before forming supported lipid bilayers on the glass. I’ve always found plasma treatment to be simpler and more consistent that chemical methods such as piranha.

2014-12-18 11.45.47

You can see a lot of single-molecule level fluorescent impurities on the glass surface before cleaning (these are a few frames stitched together):

not cleaned

And after 4 minutes of plasma treatment (with air as the process gas) it was so clean that I had trouble finding the correct focal plane:

plasma cleaned for 4 min

People are also using this plasma cleaner to treat material for PDMS bonding to glass. They say it’s been working very consistently.

So I highly recommend plasma cleaning. It takes literally a few minutes and there’s no hazardous waste to dispose of. The only real drawback is the price: a new cleaner plus pump costs several thousand dollars. In the long run, if we can get consistent science and no haz waste disposal costs, that price will be worth it. (We also split the cost with several other labs on our floor.)

I’ve also heard good things about ozone treatment. Anyone have any comments about ozone vs plasma?

Pro:

  • Very easy to use
  • Fast (<5 min) cleaning
  • Effective
  • Consistent
  • Updated models of Harrick cleaners have a nice hinged door

Con:

  • Expensive
  • Using process gases other than simply air (such as argon) is slightly more complicated, because you’ll need a tank and tubing; oxygen plasma cleaning requires a more expensive pump

comparing Nikon immersion oils

August 27, 2014 at 2:53 pm | | everyday science, review

UPDATE: update on Nikon objective immersion oils

I typically use Nikon type NF immersion oil. But I hate the dropper that it comes in, and I’ve recently been having trouble with the oil crystallizing, especially if I aliquot it to smaller dropper vials. So I decided to compare the different oil types available, namely Nikon A (not to be confused with Cargille A), Cargille B, Cargille 37, and Nikon NF. (Type 37 is sometimes called type B 37.) Note that types B and 37 are actually Cargille part numbers 16484 and 16237, respectively.

A B 37 NF montage

See full slide deck here.

My conclusion: Use Nikon A for routine imaging (the dropper is much easier to use and it’s less stinky than NF). For samples at 37 C or single-molecule imaging, use type NF.

UPDATE: Unfortunately, Nikon has discontinued their type A and NF oils. Back to the drawing board. I will update with what oil I chose to use in the future

Coherent Obis Galaxy review

August 1, 2014 at 10:50 am | | hardware, review

We recently purchased new lasers for our TIRF scope. I wanted the flexibility and low cost of a home-built laser combiner, but also I wanted the ease and stability of a turn-key laser box. I stumbled upon Coherent’s Obis Galaxy combiner, which uses up to 8 fiber pigtailed lasers and combines the emission into one output fiber. What I really love about the idea is that you can add lasers in the future as your experimental needs grow. (Or your budget does.)

The other aspect I love is that it’s just plug and play! If I were on vacation when a new laser arrived, anyone in lab should be able to add it to this system.

2014-07-03 15.22.03

We also purchased the LaserBox, which supplies power, cooling, and separate digital/analog control to 5 lasers.

2014-07-03 15.07.33

The new system just sits on the shelf. It’s tiny:

2014-07-03 15.45.57

Here it is in action. The lasers were being triggered and sequenced by the camera and an ESio board, so they were running so fast I had to jiggle my iPhone in order to see the different colors.

One problem that I have faced is that the throughput is lower than spec (should be 60%+, and it’s down at 40%). Coherent is going to repair or replace the unit. And fortunately, we’re only running the lasers at 10% or less for most experiments currently, so there’s no rush to get the throughput higher! (Edit: Coherent immediately replaced the unit and it’s now up to the correct throughput.)

If you’re ever in Genentech Hall UCSF and want a quick demo, drop me a line!

Pro:

  • Flexibility to add laser lines or upgrade lasers in the future at no additional cost (besides the pigtailed laser itself) and no downtime
  • Super easy installation
  • Cheaper than many of the other turn-key boxes
  • No aligning or maintenance needed
  • Each laser can be separately triggered and modulated (digital and/or analog)
  • Replaceable output fiber if it gets damaged (although it might not be as high-throughput as the original fiber)
  • Small and light, so it’s easy to find a place for it in any lab

Con:

  • No dual-fiber output option
  • Two boxes and some fibers going between the two makes it a little less portable than some of the other small boxes
  • No space to add optics (e.g. polarizers) in launch
  • Fans for LaserBox are not silent
  • Power and emission LEDs are too bright
  • NA of Coherent fiber is slightly smaller than that of Nikon TIRF illuminator expects, but the effect is barely observable (Coherent is working on a second fiber option that would even better match the TIRF illuminator)

Bottom line: I’d definitely recommend the Galaxy if you’re primary goals are color flexibility and simplicity. If you want more turn-key (and probably stability, but I can’t speak to that yet), there are other boxes to consider: Spectral ILE, Vortran Versa-Lase, Toptica MLE, and so on. Also, if you needed two (or more) fiber output, the Galaxy doesn’t have that option.


Edit 11/10/2014: I’ve found one issue. The NA of the Coherent fiber is smaller (0.055) than the standard Oz Optics fiber that Nikon uses for the TIRF launch (0.11). That means that the illumination is more compact at the sample. Because the beam is Gaussian shaped, that means that the illumination is less flat (i.e. very bright in the center and darker on the edges). I’m going to try a solution using a second fiber with the correct 0.11 NA and an Oz Optics AA-300 lens style universal connector. I’ll update if this works…

Edit 3/5/15: So it turns out that the NA difference isn’t that huge. Most of the discrepancy is just a difference in the way the two manufacturers report the NA. Not only that, but in practice the NA difference makes a tiny change in the illumination area in TIRF. I wouldn’t let the different NA stop you from considering this product. Also, Coherent is working on second fiber option that would even better match the TIRF illuminator. Contact Coherent to see if they can make one with a 0.074 NA (1/e2) output to match the NA of the Oz Optics (Corning) fibers that the Nikon illuminator expects.

Edit 7/30/15: The LaserBox has a 50 Ohm impedance for the digital modulation (2 kOhm for analog), because it needs to be able to driven up to 150 MHz, according to Coherent. This makes controlling the digital TTL with an Arduino a challenge, because the Uno is rated for 40 mA max. The ESio board (and maybe the TriggerScope?) can handle the higher currents. That said, the Arduino Uno seems to handle the higher current draw even though it’s not spec’ed to: I have a lot of anecdotal evidence that you can use an Arduino to control Obis lasers. (Maybe not 2 lines simultaneously?) Contact Coherent before you order to see if they can send one with higher impedance for the TTLs.

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