LED Illumination for Fluorescence


Figure 4: (A) Phase-contrast and (B–D) ratiometric pseudo-color images of Fura-2-loaded DRG sensory neurons. (B, C) Low capsaicin (30 nM, TRPV1 agonist) elicited calcium responses in sensory neurons, while (D) high capsaicin (1 μM) elicited larger calcium responses in sensory neurons.


Sensory neuron cultures were prepared from adult rat dor-


sal root ganglia and plated on a monolayer of astrocytes grow- ing on PDL-coated glass imaging dishes (MatTek). Calcium imaging was performed on Fura-2 AM-loaded cells at five days in vitro. Images were acquired with an Olympus IX73 inverted microscope with the X-Cite NOVEM and Hamamatsu ORCA Fusion BT camera. MetaFluor imaging soſtware was used for data acquisition and analysis. Adult rat DRG neuron cultured cells were loaded with 3 μM Fura-2 AM in 0.025% Pluronic F-127. Capsaicin was used to evoke calcium signals by activat- ing native TRPV1 receptors (Figures 4 and 5). Case study 3: Glutamate Elicits Calcium Fluxes in Rat


Pyramidal Neurons. Dong Liu, Matt Reissman, Mahsa Sadeghi, Adina Hazan, and Robert Petroski, Neuroservice USA (unpub- lished data). Neuron cultures were prepared from E18 rat cortex and


plated on a monolayer of astrocytes growing on PDL-coated glass imaging dishes (MatTek). Calcium imaging was performed in Fura-2AM-loaded cells at 9 days in vitro (Figures 6 and 7). Case study 4: NMDA Elicits Calcium Fluxes in Rat Cortical


Neurons. Dong Liu, Matt Reissman, Mahsa Sadeghi, Adina Hazan, and Robert Petroski, Neuroservice USA (unpublished data). Neuron cultures were prepared from embryonic day 18 (E18)


rat cortex and plated on a monolayer of astrocytes growing on PDL-coated glass imaging dishes (MatTek). Calcium imaging was performed in Fura-2AM-loaded cells at 13 days in vitro ( Figures 8 and 9). Te cells were loaded with 3 μM Fura-2AM for 30 min- utes at 37° and cells rinsed for 30–90 minutes before imaging. MetaFluor imaging soſtware was used to rapidly switch between 340 nm and 380 nm excitation wavelengths, and image pairs were acquired at 515 nm emission every 6 seconds at 390 nm. Test solu- tions were applied to the recording chamber by manual pipette.


The Green Gap Manufacturers are challenged by designing fluorescence


illumination systems that cover the same spectrum as the tra- ditionally used mercury arc lamp to adequately excite common fluorophores used in fluorescence studies. Te challenging wave- length band is between 540–590 nm, known as the “Green Gap.” LEDs in this region of the spectrum are fundamentally limited by the lack of semiconductor materials to efficiently emit light at this wavelength. Some manufacturers have generated innovative solutions to bridge the gap, including LED arrays, wavelength conversion phosphor technology, and laser solutions.


2022 May • www.microscopy-today.com


Figure 6: (A) Low resting baseline calcium level (blue) in rat cortical pyramidal neurons and the response to (B) 100 μM glutamate that triggers a rapid calcium flux (green).


Te Solution Wavelength conversion using phosphor materials


has been used in the lighting industry for a long time. Shorter wave- length light (blue, violet, UV) is absorbed by a material that re-emits


27


Figure 5: Time course of calcium responses in individual Fura-2AM-loaded DRG sensory neurons indicated by colored lines. Following a 1-minute baseline period, calcium responses of sensory neurons were detected in a low (30 nM) then high (1 μM) concentration of capsaicin for 2 minutes each. All cells responded to depolarization by 60 mM KCl, indicating healthy, electrically excitable neurons.


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