LED Illumination for Fluorescence
thousands of hours without replacement. Having a powerful, fast-switching light source providing filtered LED excitation negates the need for reliance on a filter wheel, thereby speed- ing up data acquisition. Here we demonstrate how the X-Cite NOVEM allows researchers to exploit the benefits of Fura-2 imaging (quantitation and ability to correct for bleaching) without the need for arc lamps while providing the speed and economy of use of single-wavelength probes. Te near-infrared (NIR) version of NOVEM allows imag-
ing in the NIR fluorescence region. Research interest in this this region (using indocyanine green (ICG) and IR800) is growing because of its potential use in image-guided surgery [4]. IR800 has high water solubility and stability, low toxicity, and high signal in human tumor cells due to low background autoflu- orescence in the NIR region. NIR wavelengths also allow for greater depth penetration in thicker tissues and living animals.
Figure 1: The X-Cite NOVEM, winner of a 2021 Microscopy Today Innovation Award.
Examples of Imaging with the NOVEM X-Cite LED Technology Below are some case studies describing the use of the
Table 1: Fluorophores excited by the X-Cite NOVEM with suggested filters for each wavelength.
NOVEM in calcium imaging using Fura-2 ratiometric dye. Te laboratories have traditionally used xenon lamps in their past research and have generously given their time to test the X-Cite NOVEM for similar work. Case study 1: Fura-2 Calcium Imaging of HeLa cells Using
Ionomycin. Callen T. Wallace, Senior Research Specialist, Center for Biologic Imaging, University of Pittsburgh (unpublished data). Images were acquired on a Nikon Ti inverted microscope
with a 40 × 1.30 N.A. objective and a Photometrics Prime BSI sCMOS camera using the X-Cite NOVEM light source ( Figure 3). HeLa cells were loaded with Fura-2 AM at 5 μM concentration for 30 minutes before imaging. Time-lapse imaging was performed, triggering between 340 nm and 385 nm excitation at 100 ms intervals, with a continuous acquisition to capture baseline fluorescence in each channel (Figure 3A) and the ratiometric change due to agonist (Figure 3B). To induce calcium flux, 1 mM of ionomycin was added to 2 mL of media in real-time during imaging. Case study 2: Capsaicin Elicits Calcium Responses in
Rat DRG Sensory Neurons. Dong Liu, Matt Reissman, Mahsa Sadeghi, Adina Hazan, and Robert Petroski, Neuroservice USA (unpublished data).
Figure 2: Acoustic noise comparison of the X-Cite 120Q, Xylis, and NOVEM lamp systems. The new NOVEM, even when operated at 100%, has lower noise levels.
excitation intensities. Tis alleviates the need for using cum- bersome xenon lamp technology for this application, which requires the user to align and replace bulbs every few hun- dred hours. LED technology can be used reliably for tens of
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Figure 3: Images were acquired using triggered acquisition with the light source at 100 ms exposure for each channel. The ratiometric shift occurs instantaneously on the addition of Ionomycin. (A) Pre-stimulation and (B) post- stimulation.
www.microscopy-today.com • 2022 May
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