NetNotes
4 years old and when we did the tender we compared the same slide set across several platforms (Olympus, Leica, 3D Histech, Zeiss, Hamamatsu) and felt that the Olympus came out on top at that time based on pure image quality (it is a camera rather than line scan- ner) as well as other factors. Being based on a microscope and a robot loader it also allows you to exploit microscope features that are less likely to be possible on an optical box system - e.g., overview of non- chromatically stained slides in phase contrast, darkfield, with con- denser iris closed down, on monochrome or color camera, etc. It can do single Z, EFI, virtual Z, and max projections, all with Z offset for the different fluors. We regularly run our BF dotslides in polarizing mode having identified the need from our user base, added polarizers and worked out how to get it to work - something likely not possible on an optic box. We just tested 3-channel fluor and EFI / Max / vir- tual Z on a VS110 on dried down suspensions of ciliated cells. At ×40 it clearly picks up individual broken off cilia. On the downside, being camera based, it is slower than a line scanner, soſtware is very flexible (a good thing for experienced users but therefore less good for the casual user - though it does talk you through the scan setup), it physi- cally liſts slides from rack to stage on a little finger with 2 very small vacuum suction pads and if slides are not absolutely clean, it can drop them quite readily. One other thing to consider is how the fluorescence works -
because you can’t fast scan multiple fluors by moving between filter cubes (too much heavy metal to move fast). Ours has a static quad band dichroic and fast excitation filter wheel. Te quad band is as good, bright and efficient as 4 single dedicated cubes would be, so you do need to try to balance signals across your fluors to minimize spectral bleed through on a monochrome fluor camera. One other thing to consider is how long the system has been on
the market. Longer means well established and bugs fixed, but on our dotslides, one of the cameras (Olympus CC12) has died and Olympus / OSIS won’t support it anymore so we can’t get it repaired so that sys- tem is essentially useless without shelling out for a next generation camera at considerable cost. So make sure you find out how long any system you buy will be supported for and get it in writing in any tender / purchase contract. Dave Johnston
daj1u06@soton.ac.uk
I have been looking into the Polaris, and was wondering if you
could elaborate on your experiences with it. I believe the current rea- son we have a lab interested in upgrading from the Vectra3 is speed (Polaris claims 7 colors in 20 minutes, though for a small square), but I also noticed that it claimed separation of 9 channels. Have you had any issues with the color separation? I noticed that for the Vectra fields of view on InForm, the color separation could be done tile by tile, or run across a whole slide in the same way. I find issues with either method. Unmixing is tricky business, and I’m wondering if the Polaris system has made any improvements there. Most impor- tantly, I do like my flexibility, and tend to “get creative” with what can be done on the various confocal systems we have. What limita- tions does the Polaris have that made you consider it less flexible? I too was impressed with the 3D Histech systems (speed was
great for both BF and IF on the 250), though I did not like the vari- able exposure/contrasting in the BF images. I could see in my QuPath analysis pipelines where each tile edge was due to variation in the measured OD of my cells (hopefully there is a setting where this could be turned off). Also, I would recommend being very careful about the fluor images as the mrxs format has compatibility issues. So unless you want to do all of your fluor analysis within 3D Histech’s soſtware, you might be in for a bit of pain. Some notes from the open source community on that topic:
2020 March •
www.microscopy-today.com
MRXS:
https://blog.openmicroscopy.org/file-formats/community/2016/ 01/06/format-support/ More general:
https://blog.openmicroscopy.org/community/file- formats/2019/06/25/formats/ and
https://forum.image.sc/t/ome-s-position-regarding-file-formats/ 26952 Mike Nelson
msnelson@gmail.com
We are considering a combination bright-field and fluorescence
slide scanner. Tere are several to choose from, but I’m not neces- sarily looking for specific model/vendor recommendations. For those who might be interested, there’s a nice review entitled “A Practi- cal Guide to Whole Slide Imaging: A white paper from the Digital Pathology Association” (2019) DOI: 10.5858/arpa.2018-0343 Doug Cromey
dcromey@email.arizona.edu
Based on our experience make sure that the file formats created
in various measurement configurations can be opened by your spe- cific analysis soſtware. And here you can’t trust the companies, you should really check for yourself. If they offer a “file conversion tool,” test this carefully. As an illustration I can take an already mentioned example: Te scanners from 3D Histech are fast and produced nice images, but the file format is “problematic.” Tey offer a conversion tool but in fluorescence mode this works only for 3 channels. If you want to have 4 channels (or more) it will not work. And the native file format works with only a few soſtware programs. Csucs Gabor gabor.
csucs@scopem.ethz.ch
We scan nearly 20,000 slides/year with our Aperio bright-field
slide scanner, and are up to a thousand or so per year with fluores- cence on a Zeiss AxioScan. When replacing our older Aperio (∼3 years ago) we considered getting a single scanner that can do both brightfield and fluorescence. However, since fluorescence is MUCH more time consuming we decided to get 2 new scanners instead so that fluorescence jobs don’t tie up the scanner and delay bright-field scanning. Te Aperio AT2 with 400-slide loader gives us excellent throughput for bright-field scanning; while our Zeiss AxioScan works well for fluorescence (it can do brightfield too). So I guess my first sug- gestion would be if you are in a research hospital like me and have lots of brigh-tfield slides, consider having a dedicated bright-field scanner and then a second scanner for everything else. Note, our slide scan- ners are used by my staff only - we don’t train users to run them. Tis improves throughput and, with thousands of slides of expertise we get excellent results nearly every time. Tey also watch for overhang- ing coverslips, uncured mounting media, etc. and therefore prevent costly downtime. I know others allow users to scan themselves, but it hasn’t been our approach. Brightfield is relatively straightforward and we didn’t see a huge
distinction between the scanners that are out there, particularly if you’re looking for one with a more moderate 100-slide loader. Res- olution, speed, quality are all pretty similar. Line scanning versus camera based scanners doesn’t seem to make a big difference (despite what the companies will tell you on both sides of the debate). Per- sonally, I prefer a scanner that shows me a preview of the tissue, the region it plans to scan (based on tissue finding algorithm), and a map of focus locations. We scan only research slides (not clinical), and many research slides are pretty ugly. If we allowed the automatic tis- sue finding and focus algorithms to run, our failure rate would prob- ably be 10 to 20%. On the other hand, aſter running the preview on a batch of slides, our operators (my staff) review them and adjust the tissue area and focus points to exclude junk and this lowers the failure rate to below 1%. By failure rate, I mean that if there is a focal point
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