How Can The Latest Developments in Beam Shaping Improve Microscopy?
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| Lizenz | CC-Namensnennung 3.0 Unported: Sie dürfen das Werk bzw. den Inhalt zu jedem legalen Zweck nutzen, verändern und in unveränderter oder veränderter Form vervielfältigen, verbreiten und öffentlich zugänglich machen, sofern Sie den Namen des Autors/Rechteinhabers in der von ihm festgelegten Weise nennen. | |
| Identifikatoren | 10.21036/LTPUB10783 (DOI) | |
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00:00
StandardzelleZelle <Mikroelektronik>LäppenHerbstWooferZugmaschineMikroskopWerkzeugBesprechung/Interview
00:09
MikroskopStrahlformungZelle <Mikroelektronik>KristallgitterZelle <Mikroelektronik>KristallgitterPfadfinder <Flugzeug>TonsignalVerpackungMechanikerin
00:22
Pfadfinder <Flugzeug>Elektrisches SignalBesprechung/Interview
00:29
BeleuchtungsstärkeSondeMikroskopBesprechung/Interview
01:06
MikroskopAngeregter ZustandElektrisches SignalBeleuchtungsstärkeBesprechung/Interview
01:35
StrahlformungInfrarotlaserSchlauchkupplungNiederspannungsnetzMikroskopHalbleiterlaserBeleuchtungsstärke
01:48
NiederspannungsnetzWarmumformenOptische KohärenzGammaquantTotalreflexionProfil <Bauelement>NiederspannungsnetzAbstandsmessungWellenlängeBeleuchtungsstärkeInfrarotlaserIntensitätsverteilungOptisches SpektrumGauß-BündelSchmiedenOptische KohärenzWarmumformenErsatzteilReflexionskoeffizientDrechselnTuner
02:53
FernrohrMitsubishi LancerFeinschneidenBesprechung/Interview
03:00
WarmumformenAbstandsmessungMikroskopobjektivBeleuchtungsstärkeGauß-BündelMikroskop
03:31
Besprechung/Interview
03:42
StrahlformungMikroskopGauß-BündelBesprechung/Interview
04:03
SpektralbandeSpiegelungNiederspannungsnetzHintergrundstrahlungRauschsignalElektrisches SignalKraft-Wärme-KopplungIntensitätsverteilungOptisches InstrumentVerpackungZelle <Mikroelektronik>WarmumformenLaserLichtKlangeffektPhototechnikFärberSpeckle-InterferometrieReflexionskoeffizientOptische KohärenzLuftstromDrechselnTotalreflexionSchwellenspannungFluoreszenzmikroskopieSchmieden
06:10
Source <Elektronik>RauschsignalFärberTextilfaserOptische KohärenzHintergrundstrahlungGauß-BündelBeleuchtungsstärkeLaserDrehmasseBesprechung/Interview
06:38
StrahlformungElektrisches SignalBrennstoffClosed Loop IdentificationSpeckle-InterferometrieMikroskopErsatzteilFernordnungNahtBesprechung/Interview
07:07
PaketvermittlungProzessleittechnikProfil <Bauelement>BeleuchtungsstärkeFernordnungKraft-Wärme-KopplungZylinderkopfSchnittmusterErsatzteilSondeSpeckle-InterferometrieBesprechung/Interview
07:58
StrahlformungKalenderjahrRotationszustandSpeckle-InterferometrieBeleuchtungsstärkePropellergondel
08:10
Ladungsgekoppeltes BauelementDrechselnBesprechung/Interview
08:31
Spezifisches GewichtTotalreflexionBeleuchtungsstärkeReflexionskoeffizientBesprechung/Interview
08:46
Speckle-InterferometrieSchwächungThermostatBesprechung/Interview
09:14
Computeranimation
Transkript: Englisch(automatisch erzeugt)
00:04
Microscopes are one of the standard tools in almost every lab nowadays. It gives us the ultimate chance to look at living cell interaction, to look at sub-cell structures and actually learn a lot about biochemics there.
00:22
One special research field in the microscopy section is the fluorescence labeled microscopy. There you have the big challenge that you're depending on your fluorescence signal for gathering information. You have to be really sure that if you get an uneven response from your spasm
00:46
that this uneven response is actually resulting from an uneven or inhomogeneous response from your probe and not coming from an uneven illumination and therefore interfering with your non-linear interaction with the fluorescence molecules.
01:06
Therefore our question, our research question is how and when can we create an illumination for such microscopic applications that are so homogeneous that you can be definitely sure that the illumination is not messing with your results
01:24
and that you're looking at the pure interaction of your excitation and then your fluorescence signal that's coming back from your spasm. In a common microscope on this area, in the fluorescence microscopy,
01:44
usually lasers or laser diodes are used for illumination. Therefore either they are fiber-coupled or free-beam lasers but they have a Gaussian beam distribution. It's very bright in the center and the intensity declines to the edges.
02:01
This is definitely not something we want. And additionally because we're dealing with that special total internal reflection fluorescence microscopy, we have to address other attributes as well. So we need a high spatial coherence to make that microscopy method work.
02:21
We have to have a long working distance which means the distance between where the profile is originally generated and where the profile then is used has to be several centimeters. It has to be achromatic so it has to work for all the wavelength in the visible region at the same time.
02:42
And of course it has to have a high throughput efficiency. We do not want to lose any light, any photon in between in that illumination path. For all those reasons we choose to go for a setup that almost looks like a Galilean telescope.
03:01
So it's made out of two lenses, two very special lenses. It's aspheric lenses and those lenses are facing each other and they're calculated in a way that the rays that come from the first surface that are redistributed to achieve this homogeneous illumination
03:22
are collected by the second lens and then recollimate this beam to go for this long working distance. By doing this approach we actually were able to achieve all the requirements to work within such a microscope setup.
03:45
Now with that optical design being done for the beam shaping device we actually built that and then it was implemented into a microscope setup to actually figure out how much of the demands we could actually meet in experimental testing.
04:03
So the first thing we looked at was the threshold. By illuminating your spasm, either with the Gaussian distribution or with a top hat distribution it's essential to know how much light you give into the system to get your fluorescence signal at an optimum so you can actually work with the data.
04:25
For the Gaussian distribution it's extremely sensitive to not overdo it in the center and actually have enough light on the edges to get a signal as a response. For the top hat distribution we could find that there's a huge region actually
04:43
where we could change the incoming intensity and still get a stable counting of labeled cells in that experimental work. The second thing we looked at was photobleaching. Of course fluorescence dyes have a photobleaching effect.
05:01
This is just physics at that point. You excite the dye, the dye is giving the light back and at a certain time it's losing that ability and then it's just going dark at that certain spot. That's not problematic because we're talking about seconds so you have enough time to get your image
05:20
but it gets problematic if the bleaching is not homogeneous especially if you have a spasm that has dynamics and you're looking into fluid interaction or something. So what we could find is that we have an extremely homogeneous photobleaching time all over the illuminated area having that top hat distribution.
05:44
The third thing we looked into was the background noise. If you do total internal reflection fluorescence microscopy it's all about just illuminating the top layer so you're actually just looking at the top cells of your spasm
06:01
and everything you get from the volume behind is more or less a background noise messing up your fluorescence signal. For achieving that you need that high spatial coherence and we could show that with our optics or with a beam shaping device we could keep the high spatial coherence from our illumination source from the laser dye
06:25
and get that through the complete system and reduce the background noise by a significant factor compared to having a multimode fiber or something like that for illumination.
06:42
In the experiment I just described it's giving you better results. You have the better reliability that your fluorescence signal is actually coming from regions that want to interact with you and if you don't see anything then probably there's nothing there. What's even more interesting or what new options you have for that
07:03
is going into high throughput stitching. Since you only can see a small part of your spasm when you look through a microscope, something like maybe 30 microns in diameter then you have to put images together in order to see a whole picture.
07:22
To do so you have to move your spasm and you have to take several frames. If you have an even illumination like we demonstrated with the top head profile you have the chance to do that without further data processing and not ending up with a checkerboard pattern
07:41
where you don't really know if it's coming from the illumination or actually there is parts missing in your sample. I think there's a huge chance of speeding up optimized high throughput biological probe imaging there.
08:02
I think there are two main topics here. The first thing especially for the high throughput imaging would be to lose that rotational symmetry illumination path and actually go for a square or a rectangular shape because your CCD camera has a rectangular shape usually
08:21
and also that stitching is more efficient and can even speed up more if you add rectangles next to each other. The second thing is of course right now we were limiting ourselves just to one very specific field in microscopy. So we were just looking at this total internal reflection for sense microscopy.
08:46
Of course that approach to go for homogeneous illumination can be expanded to other fields in microscopy. Even common microscopy where you just look through and see an image of whatever you want to look at
09:01
can benefit because you see a more homogeneous illuminated image there but also more advanced techniques like STAT microscopy can benefit from this approach.