Equipment
Combined Spinning Disk and Widefield Fluorescence Microscopy setup: “Schneewittchen”
The Nikon Eclipse TI: HUBC/A inverted microscope is a setup for bright field and fluorescence widefield and multi-confocal imaging. The entire microscope and its atmosphere can be thermostated and the temperature can be controlled by an Okolab incubation chamber powered with an Okolab temperature controller allowing for a precise control of the temperature from room temperature up to 50 °C. Air humidity and protection atmospheres can also be controlled.
Different sample holders in a combination with several objectives allow for a wide range of samples to be investigated.
For excitation different laser can be used:
- 405 nm: Toptica iBeam smart 405S-HP 300 mW
- 488 nm: Toptica iBeam smart 488S-HP 200 mW
- 561 nm: Cobolt Jive TEM 500 mW
- 640 nm: 2x Toptica iBeam smart 640 CD 200 mW with different beam polarization
Laserpower can be enhanced using a Lumencor Spectra III light engine with different LEDs:
- 390/22 nm at 500 mW
- 440/20 nm at 500 mW
- 475/28 nm at 500 mW
- 510/25 nm at 400 mW
- 555/28 nm at 500 mW
- 575/25 nm at 500 mW
- 635/22 nm at 500 mW
- 747/11 nm at 500 mW
The collection of objectives is available for the different measurement demands:
- Olympus UPlanSApo 60x/1.20 Water
- Nikon S Plan Fluor ELWD 40x/0.60
- Nikon HP Plan Apo VC 100x/1.40 Oil
- Nikon Apo TIRF 100x/1.49 Oil
The setup also includes a Yokogawa CSU-X1FW spinning disk unit with a spinning frequency up to 5000 rpm. A beamsplitter Di01-T405/488/568/647 quad-band dichroic mirror incl. filter covers a wide range of the visible spectrum to apply fast confocal microscopy techniques. The image will be captured using an Andor iXon Ultra EMCCD (1024 x 1024 pixel, 26 fps, Electron Multiplying (EM) mode). Further filters for optional use in the spinning disc filter wheel to reduce unwanted signals are:
- Chroma ET 455/50m Bandpass
- Chroma ET 525/50m Bandpass
- Chroma ET 605/70m Bandpass
- Chroma ET 685/70m Bandpass
- Chroma ET 700/75m Bandpass
- Semrock 617/73 Brightline HC Bandpass
For 3D super-resolution fluorescence microscopy (3D-SRFM) techniques, a Double Helix Optics Spindle[1] module is implemented in combination with a Photometrics Prime 95B CMOS Camera (1,44 MP, 41 FPS 16bit, 80 FPS 12bit) within a second emission path. SRFM images with a resolution far beyond the diffraction limit can be generated. Two Double Helix phase masks are available covering the emission wavelength of around 580 nm and 670 nm.
Using this setup offers the possibility for a non-invasive microscopy with a spatial resolution below 50 nm for all 3 dimensions and even below 25 nm for the xy-plane.[2] Using the Double-Helix phase mask to modify the point spread function will lead in information of the third spatial dimension even in a 2D image.
Literature
[1] S. R. Pavani, R. Piestun, Opt Express 2008, 16, 3484-3489.
[2] G. Grover, S. Quirin, C. Fiedler, R. Piestun, Biomed Opt Express 2011, 2, 3010-3020.
Olympus IX83 inverted fluorescence microscope: “Hänsel”
Widefield (Köhler) and TIRF illumination, as well as a confocal imaging branch, is available. The sample can placed on a coverslip or into a liquid sample holder. It can be moved with a xyz-piezo table (P-545.3R7, Physik Instrumente). We possess different devices to cool and heat the samples in a range of 8 °C up to 200 °C (ITO cover glass). Furthermore, we can adjust also the humidity via a moisture chamber.
The following objectives are available
Olympus 60XO UPLSAPO (NA:1.35; WD: 0.15 mm) oil
Olympus 100XO2 UPFLN (NA:1.3; WD: 0.2 mm) oil
Olympus 60XW UPLSAPO (NA1.2; WD: 0.28 mm) water
Olympus 100XO UApoN (TIRF) (NA: 1.49) oil
Various filters are available in the filter wheel
Pos 1: DC: ZT 458 RDC (Chroma)
Pos2: DC: ZT 488 RDC (Chroma)
Pos3: DC: HC BS R514 (Semrock)
Pos4: DC: "Triple Line" (ZT 405/561/657rpc-UF1 dichroic) (Chroma)
Pos5: DC: "TIRF Quad-Line" (ZT 405/488/561/640rpc) (Chroma)
Pos6: DC: ZT 405/488/532/640 (Chroma)
Pos7: DC: ZT 561 (Chroma)
A set of different emission filters are available. The most often used ones are:
ET 510 LP (Chroma)
ET 685/70 M (Chroma)
ET 500 LP (Chroma)
617/73 BrightLine HC (Semrock)
ZET405/473/532/640m (Chroma)
For excitation, the following lasers can be used
378 nm diode laser (i beam smart 375-S Toptica, 70 mW)
488 nm ultra-compact diode laser (Cobolt MLD, 200 mW)
532 nm (Laser World, 500 mW)
561 nm diode pumped solid state laser (Cobolt Jive, 200 mW)
638 nm ultra-compact diode laser (Cobolt MLD, 150 mW)
Argon Laser (American Laser Corporation)
Detection path
The emission signal can then be separated by wavelength or by polarization in two channels by an Optosplit II bypass (CAIRN Research). Currently, the device includes a ZT 594 RDC dichroic filter (AHF Analysentechnik) to split the emitted light in two channels (< 594 nm and > 594 nm). Additionally, further emission filters can be implemented via this device.
Before the signals will reach the chip of the EMCCD camera they will pass through a cylindrical lens (focal length 500 mm). Via this an astigmatism is induced so that the z-position of the emitters can be determined.[1]
The light of the two Optosplit channels is detected by an EMCCD Camera (512x512 pixels, Andor IXon Ultra 897) on two different regions of the chip. The resulting pixel size after magnification of the setup is typically 80 nm per pixel.
With this setup we are able to perform super-resolution fluorescence microscopy. Furthermore, via the Optosplit module we are able to split the emitted light by its wavelength in two different channels and by this image it on two different regions of the EMCCD chip. Using the solvatochromic dye Nile Red, Purohit et al. visualized the polarity of microgels on a nanoscopic scale.[2]
Literature
[1] B. Huang, W. Wang, M. Bates, X. Zhuang, Science
2008, 319, 810.
[2] A. Purohit, S. P. Centeno, S. K. Wypysek, W. Richtering, D. Wöll, Chemical Science 2019, 10, 10336-10342.