On 8/08/2024 3:25 am, John Larkin wrote:
On Wed, 7 Aug 2024 16:22:05 -0000 (UTC), Phil Hobbs <[email protected]> wrote:
John Larkin <[email protected]> wrote:
https://www.planetanalog.com/wp-content/uploads/Fig-1-STED-precise-temp-measurement.png?resize=1260,709
Brilliant soldering job, for sure. :(
Cheers
Phil Hobbs
I like the fingerprints on the Scotch tape, and the hairball on the
probe. And all the dust.
None of which matters.
Stimulate emission depletion (STED) microscopy is not a mode of
temperature measurement that I had heard of, and John Larkin is likely
to be just as ignorant, but rather than finding out what is actually
going on he merely sneers about the unpretentious image.
Google managed to find this for me.
"Stimulated emission depletion (STED) microscopy delivers resolutions
better than 20 nm. Due to significant progress in STED instrumentation
and the commercialization of new, photo-stable dyes, STED is now widely accessible. However, although STED offers unlimited resolution in
theory, photobleaching comes into play in practice. Mediating its
effects can require tuning down the resolution or recording fewer time
steps and smaller 3D-volumes than desired. Besides Adaptive
Illumination, a solution to circumvent the physical limitations of
current labeling technology is the adaptation of the PAINT-concept
(point accumulation for imaging in nanoscale topography) to STED
microscopy. PAINT is based on the application of exchangeable labels
that only temporarily bind to their target structures. During
acquisition, these labels are constantly replenished from a large pool
in the imaging medium, providing stable sample brightness even at the
highest resolutions.
The resolution of a STED microscope scales with the intensity of the
STED laser. Unfortunately, an increase in resolution is therefore often connected to an increase in photo-bleaching. The available fluorescence
photon budget thus can hinder time lapses as well as volume imaging.
This is because with time lapse imaging, the sample area is illuminated multiple times and the number of fluorophores is concomitantly reduced
each time. Similarly, when imaging volumes, adjacent z-planes are imaged
in succession, so that each plane is subjected to multiple rounds of illumination and therefore photobleached to a certain extent each time.
As a result, microscopists often need to sacrifice either resolution,
the number of time steps or the number of z-planes recorded in order to
reduce photobleaching to an acceptable level.
The situation described above was particularly true for early
implementations of STED microscopy, when very strong sample illumination
met a small number of photo-stable fluorophores. Today, major
developments in STED instrumentation, such as the use of optimized
pulsed STED lasers 1, highly efficient detection via APDs, and Adaptive Illumination 2, 3, 4 now allow a strong reduction of sample
illumination. Currently, state-of-the-art STED microscopes readily offer resolutions reaching <20 nm and allow the acquisition of live cell
movies and image stacks. At the same time, a vast range of optimized,
bright, and photostable fluorophores for STED imaging is now
commercially available and comes with many conjugation chemistries for
diverse applications 5-9.
Nevertheless, the achievable resolution and number of image frames are
still ultimately limited by photobleaching, albeit at a much higher
level than only a few years ago.
On the other hand, PAINT allows to suppress or bypass the photobleaching process and therefore offers additional resolution and extended
time-lapse or volume imaging. With conventional PAINT microscopy,
individual, transiently binding dyes are located precisely using single molecule localization microscopy 10, 11. Most importantly, fluorophores
are constantly exchanged: bleached fluorophores are replaced with fresh
ones from the large reservoir of the imaging medium using labels with
transient binding properties. This allows for fast and continuous
exchange of fluorophores during image acquisition.
Recently, Spahn et al. 12 have successfully adopted exchangeable
fluorophores for use with STED microscopy. In contrast to classical
PAINT, which requires very few dyes bound at a single time, staining for
STED was optimized to provide high densities of bound dyes while
allowing fast replenishment from the imaging medium. Interestingly, this
can be implemented with a large range of staining techniques, including
simple lipophilic dyes, DNA stains, toxin- and peptide-conjugated dyes
as well as with immunostaining with DNA-labeled antibodies."
Bizarre stuff, but no doubt fascinating to some.
--
Bill Sloman, Sydney
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