Instrumentation
A. New Technologies
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(a) Benign multiphoton UV imaging:
Our technological innovations in the area of multiphoton UV microscopy have attracted a number of biologists, with whom we have collaborated to make key discoveries. Our non-epifluorescent OPO based dopamine imaging technology is perhaps the deepest ultraviolet live cell imaging instrument available at present. While this has been utilized for imaging neurotransmitters so far, it is possible that other uses of this new slice of optical spectrum will become apparent. |
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(b) Fiber FCS:
We have also developed a fiber-based FCS technique, which in principle can perform FCS in vivo and can detect soluble aggregates [Garai et al., 2006]. This, together with our discovery of spontaneous protein aggregation on quantum dots [Sahoo et al., Chem. Phys. Lett., 2007], is an important advance towards the in vivo detection of toxic species such as misfolded proteins.
We have also developed a fiber-based FCS technique, which in principle can perform FCS in vivo and can detect soluble aggregates [Garai et al., 2006]. This, together with our discovery of spontaneous protein aggregation on quantum dots [Sahoo et al., Chem. Phys. Lett., 2007], is an important advance towards the in vivo detection of toxic species such as misfolded proteins.
(c) A spatial pH jump technique for measuring chemical kinetics:
We demonstrated a spatial pH jump technique that can measure the kinetics of a pH induced chemical reaction in the steady state. The pH jump is created by the multiphoton excitation of a photo-excitable proton donor. This creates a small open volume of low pH in a solution. When the reactant molecules diffuse into this volume, they undergo a reversible chemical change. The fluorescence lifetime of these molecules bear the signature of this chemical change. We have demonstrated the technique by creating a spatial pH jump using o-NBA and by measuring the changes in the lifetime of fluorescein within the first few milliseconds of entering this low pH region. [Nag et al., 2009]
We demonstrated a spatial pH jump technique that can measure the kinetics of a pH induced chemical reaction in the steady state. The pH jump is created by the multiphoton excitation of a photo-excitable proton donor. This creates a small open volume of low pH in a solution. When the reactant molecules diffuse into this volume, they undergo a reversible chemical change. The fluorescence lifetime of these molecules bear the signature of this chemical change. We have demonstrated the technique by creating a spatial pH jump using o-NBA and by measuring the changes in the lifetime of fluorescein within the first few milliseconds of entering this low pH region. [Nag et al., 2009]
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(d) A compact FCS spectrometer with a foldable light-path:
We have now built a miniaturized, alignment-less fluorescence correlation spectrometer. This spectrometer is a modification of our all-fiber design, which was adequate for detecting protein aggregates, but not sensitive enough to detect single dye molecules. Our current design uses a high NA objective mated to an all-fiber spectrometer design, in a way that increases sensitivity to single-molecule levels. The optical part of the resulting device is about 18x4x4 cm3 , which is about two orders of magnitude smaller in volume compared to a typical FCS spectrometer. The rest of the machinery is connected through flexible electrical and fiber optic cables, and can be separately packaged in a small palm-top box. This device brings the possibility of making a field deployable and affordable FCS instrument, which can be important for clinical diagnostic applications. |
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B. Other in-house developments
(a) An AFM-Confocal-FCS setup:
We have now built an AFM-Confocal-FCS setup. In this setup, the Center of the ~250 nm optical excitation spot and the AFM cantilever tip (~30 nm radius) can be brought together with in 100 nm of each other very precisely, such that it can perform fluorescence confocal microscopy and AFM imaging simultaneously for the same field by stage scanning. This instrument can also be used for two dimensional membrane FCS and three dimensional FCS experiments.
This is an excellent instrument for understanding protein-lipid (membrane) interactions.
(a) An AFM-Confocal-FCS setup:
We have now built an AFM-Confocal-FCS setup. In this setup, the Center of the ~250 nm optical excitation spot and the AFM cantilever tip (~30 nm radius) can be brought together with in 100 nm of each other very precisely, such that it can perform fluorescence confocal microscopy and AFM imaging simultaneously for the same field by stage scanning. This instrument can also be used for two dimensional membrane FCS and three dimensional FCS experiments.
This is an excellent instrument for understanding protein-lipid (membrane) interactions.
(b) A Total Internal Reflectance Microscope:
We have built a TIRF microscope using a special high numerical aperture objective. The TIRF approach angle can be adjusted continuously over a range, which enables different depth resolutions. We have used it to follow vesicle release dynamics in primary cultured neurons with a time scale of ~ 10 ms.
We have built a TIRF microscope using a special high numerical aperture objective. The TIRF approach angle can be adjusted continuously over a range, which enables different depth resolutions. We have used it to follow vesicle release dynamics in primary cultured neurons with a time scale of ~ 10 ms.
(c) Lifetime determination in small volumes:
We have built a multiphoton excited micro-TCSPC (time correlated single photon counting) set-up for interrogating protein aggregation. The instrument has an instrument response function of about 65 ps (without deconvolution). It works with the reverse start stop mode and can average at a rate of 76 MHz for fast decaying samples. The instrument can work with as little as 10 microliters of solution, and in this solution, the interrogated volume is only about 1 femtoliter. This instrument should be suitable for measuring lifetime of fluorophores inside cells.
We have built a multiphoton excited micro-TCSPC (time correlated single photon counting) set-up for interrogating protein aggregation. The instrument has an instrument response function of about 65 ps (without deconvolution). It works with the reverse start stop mode and can average at a rate of 76 MHz for fast decaying samples. The instrument can work with as little as 10 microliters of solution, and in this solution, the interrogated volume is only about 1 femtoliter. This instrument should be suitable for measuring lifetime of fluorophores inside cells.
(d) A facility for the synthesis and purification of peptides:
To understand the aggregation of peptides such as amyloid beta, it I important to be able to site specifically mutate the amino acids. To achieve this with full flexibility, we have installed a peptide synthesizer and an HPLC machine. These two, together with the analytical mass-spectroscopic facilities already available in the department, have enabled us to purify peptides such as amyloid beta (40 residues) and many of its variants and fragments. These fragments and variants will allow us to understand the role of specific types of amino acids in the aggregation process. We are also producing isotopically labeled peptides suitable for solid state NMR studies.
To understand the aggregation of peptides such as amyloid beta, it I important to be able to site specifically mutate the amino acids. To achieve this with full flexibility, we have installed a peptide synthesizer and an HPLC machine. These two, together with the analytical mass-spectroscopic facilities already available in the department, have enabled us to purify peptides such as amyloid beta (40 residues) and many of its variants and fragments. These fragments and variants will allow us to understand the role of specific types of amino acids in the aggregation process. We are also producing isotopically labeled peptides suitable for solid state NMR studies.
C. Dissemination of knowledge and commercialization of technologies
(a) Dissemination of technological know-how:
We have also launched projects to disseminate our technological know-how, to help bolster the culture of instrument building amongst Indian biophysicists. We conceived of and executed a one-of-a-kind workshop in 2009 where we taught researchers from many Indian institutions the art of building single-molecule spectrometers (www.fcsworkshop.in). These researchers were given a kit each from which they built fluorescence correlation spectrometers themselves under our supervision, and they were then allowed to take these spectrometers to their respective home institutions [sponsored by a grant from the Dept. of Information Technology]. This workshop was repeated this year for reviewing the progress in the different labs which received the kit, and to teach them how to add lifetime determination capabilities to the existing kit. There has been enthusiastic response from the community and this now promises to become an annual fluorescence workshop of the country, where technical knowledge is exchanges at an effective level, in addition to discussing science that is made possible by these techniques.
(a) Dissemination of technological know-how:
We have also launched projects to disseminate our technological know-how, to help bolster the culture of instrument building amongst Indian biophysicists. We conceived of and executed a one-of-a-kind workshop in 2009 where we taught researchers from many Indian institutions the art of building single-molecule spectrometers (www.fcsworkshop.in). These researchers were given a kit each from which they built fluorescence correlation spectrometers themselves under our supervision, and they were then allowed to take these spectrometers to their respective home institutions [sponsored by a grant from the Dept. of Information Technology]. This workshop was repeated this year for reviewing the progress in the different labs which received the kit, and to teach them how to add lifetime determination capabilities to the existing kit. There has been enthusiastic response from the community and this now promises to become an annual fluorescence workshop of the country, where technical knowledge is exchanges at an effective level, in addition to discussing science that is made possible by these techniques.
(b) Building a Photonics talent pool in India:
Through another grant from DIT, we have helped institute the National Photonics Fellowship of India. The main goal of the fellowship is to expose the best students in Physical sciences to high quality research and help them decide about a future career in Photonics R&D (academic or corporate). About fifteen students (soon-to-graduate or fresh graduates) would be chosen each year and given the opportunity to spend between 3-12 months in a selected laboratory in a premier academic institute in India. The student will be free to choose a laboratory from a list of laboratories who have agreed to mentor such students. They will spend their time contributing to a research project in photonics, and full financial support will be provided from the grant (www.photonicsindia.org).
Through another grant from DIT, we have helped institute the National Photonics Fellowship of India. The main goal of the fellowship is to expose the best students in Physical sciences to high quality research and help them decide about a future career in Photonics R&D (academic or corporate). About fifteen students (soon-to-graduate or fresh graduates) would be chosen each year and given the opportunity to spend between 3-12 months in a selected laboratory in a premier academic institute in India. The student will be free to choose a laboratory from a list of laboratories who have agreed to mentor such students. They will spend their time contributing to a research project in photonics, and full financial support will be provided from the grant (www.photonicsindia.org).
(c) Commercialization of technologies:
M/s Holmarc of Kochi, India is presently negotiating with DIT to make the FCS kit available commercially. M/s PicoQuant has already agreed to supply the electronics part of the kit at a discounted rate, which should make it affordable for researchers in colleges and universities.
A technology for performing sensitive FCS measurements in a confocal microscope has now been granted a US patent, and we are now in discussion with several firms about commercialization of this technology [Kaushalya et al. X]. Our developments in the area of UV microscopy have attracted M/S Zeiss MicroImaging, the leading microscopy company in the world, to sign an agreement with TIFR to co-develop these technologies. Work has started with technical collaboration and instrumental support from Zeiss MicroImaging, in return for a preferred licensing agreement about future technologies developed here. An Advanced Microscopy Research Center has been founded for this purpose, where not only our lab, but a few other TIFR academics would be working to explore their ideas about developing interesting biophotonic tools of the future.
M/s Holmarc of Kochi, India is presently negotiating with DIT to make the FCS kit available commercially. M/s PicoQuant has already agreed to supply the electronics part of the kit at a discounted rate, which should make it affordable for researchers in colleges and universities.
A technology for performing sensitive FCS measurements in a confocal microscope has now been granted a US patent, and we are now in discussion with several firms about commercialization of this technology [Kaushalya et al. X]. Our developments in the area of UV microscopy have attracted M/S Zeiss MicroImaging, the leading microscopy company in the world, to sign an agreement with TIFR to co-develop these technologies. Work has started with technical collaboration and instrumental support from Zeiss MicroImaging, in return for a preferred licensing agreement about future technologies developed here. An Advanced Microscopy Research Center has been founded for this purpose, where not only our lab, but a few other TIFR academics would be working to explore their ideas about developing interesting biophotonic tools of the future.
