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Question

Use this page as the title page; print your name on top. Staple additional pages. If formulas are used, provide them. Provide numerical answers in required units; circle the answers. What is importance of channelrhodopsin's operating in the near-infrared part of light spectrum? Why is it more difficult to alter optical properties channel rhodopsin's in comparison to GFPs? Briefly explain the effect of illumination of neurons expressing halo rhodopsin on neuron's membrane potential. What is approximately the optimal pinhole diameter (in pm) for a confocal microscope with a water-immersion objective lens 40x (NA=0.8) and a 488-nm laser? What is the approximate slice thickness (in pm) imaged by the confocal microscope above? Prove that lateral resolution is always better than axial resolution in confocal microscopy. Laser of what wavelength (in nm) should be used to image fluorescence of Ca-sensitive dye Rhoda- 2 in a 2-photon confocal microscope? Assume that 3 light beams are employed in a STED microscope with wavelength (X) values of 488 nm, 540 nm and 560 nm. Which X corresponds to the excitation beam, which X corresponds to the depletion beam, and which X corresponds to the emission beam? What is the theoretical diffraction-limited resolution (in nm) of electron microscope in which electrons are accelerated by the voltage of 50 kV? What is the cause of "chromatic" aberration in electron lens? Why samples for electron microscopy have to be dehydrated? What is the actual resolution (in nm) of electron microscope operating at accelerating voltage of 70 kV with a magnetic lens operating at aperture angle of 0.5 degree? Why samples for SEM have to be conductive? What is Relative Centrifugal Field (RCF, in g's) in a centrifuge with a rotor radius of 10 cm spinning at a rotation speed of 12,000 rpm? If centrifugation of a cell suspension at rotation speed of 1, 200 rpm takes 3 min, how much time will be required to achieve the same degree of cell sedimentation at rotation speed of 600 rpm? Assume that spherical particles with diameter = 100 pm and density=1.1 g/cm^3 have sedimentation velocity of 1 cm/min in a solution with density = 1 g/cm^3. What is the sedimentation velocity of spherical particles with diameter = 50 mu m and density = 1.3 g/cm^3 in the same conditions? Two ribosomal sub-units have sedimentation coefficients of 60S and 40S. Assuming that both subunits have spherical shapes and their densities are equal, calculate the ratio of their molecular weights, (internet search for molecular weights is not sufficient) Which method, Rate Zonal Centrifugation or Isopiestic Centrifugation, is better suited to separate RNAs from proteins? Briefly explain why.

Explanation / Answer

1. Channelrhodopsins are basically rhodopsins that function as light-gated ion channels. They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis:it means control movement by sensitization of light. it has become possible for the neuronal activity to be optically manipulated using biological photo-reactive molecules such as channelrhodopsin. it is mostly reactive to visible light, which does not effectively penetrate through biological tissues.In contrast, near-infrared light region is 660–1455nm penetrates deep into the tissues because biological systems are almost transparent to light within this, so it is called window. Using optogenetics, target neurons have been genetically modified to express light-sensitive proteins such as channelrhodopsin, which is the algel protien. it controlled to become active under irradiation by a light-emitting diode ot it is also inducted by LASER. meanwhile it has been shown with the Cl/H+ transporter rhodopsins eg. as halorhodopsin (Halo/NpHR) and archaerhodopsin (Arch/aR-3, ArchT). so it will be activated by the red light it is due to ChRs responsive to red light have been obtained through molecular modifications. so NIR light could be applied for the optogenetic manipulation of neural activities when LNPs are act as donors and ChRs act as acceptors. so by using LASER or LED it creats photosensitization of ChRs and it will become responsive to near infrared region.

2. the factor which afects on the activity of Channel conductance, Ion selectivit, Kinetics, Desensitisation and recovery from the desensitised state, Light sensitivitySpectral response, Membrane trafficking and expression The main deficiency of ChR2 is the high level of desensitisation as comare to DPFs, which reduces the current by ~79% at physiological pH it is fully recoverd within the 25 second if dark matter is be there. ChR was the first channelrhodopsin used to excite neurons with light but when it stimulated with high intensity light, ChR has a rapid on-rate and moderate channel closing rate as compare to DPFs due to that ChR is maximally excited by 470nm light only while the range of DPFs is broader. based on that The conductance of ChR is estimated to be 50–250 fSwhile for DPFs it is higher.

3. Halorhodopsin is a light-gated ion pump specific for chloride ions. It is a seven-transmembrane protein of the retinylidene protein family, homologous to the light-driven proton pump bacteriorhodopsin, and samein tertiary ructure to vertebrate rhodopsins, the pigments that sense light in the retina.

Halorhodopsin also shares sequence similarity to channelrhodopsin, another light-driven ion channel. Halorhodopsin contains the essential light-isomerizable vitamin A derivative all-trans-retinal. Due to the intense attention on solving the structure and function of this molecule.

Halorhodopsin has been used to achieve inhibition of action potentials in neurons in mammalian systems. though activation of NpHR leads to an influx of chloride ions which is a part of the natural process for generating hyperpolarization, NpHR induced inhibition works very well in neurons. Original NpHR channels when expressed in mammalian cells, showed a tendency to get accumulated in the endoplasmic reticulum of the cells.

halorhodopsin expressing transgenic cellre generated (molecular modification. Optical stimulation of the halorhodopsin expressing neurons induced a hyperpolarization on membrane potential and inhibited the firing of action potential. Attempts to control spontaneous seizures in a temporal lobe epilepsy cells achieved through optogenetics

4.  point spread function at pinhole= Airy disk diameter × magnification of lens = 1 Airy unit
= resolution of lens × magnification of lens × 2

so oil immersion llence it will be 100 but here it 40 put value it gives answe 26 micrometer

5. 70 micrometer.

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