Help with viewing bacteria

[Shane van Breda]

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Dear group members,

I have a very simple question that I require help with. Unfortunately our confocal expert has
been booked off for three months and I am struggling.

I am using a Zeiss LSM 510 Meta. I am viewing bacteria using a 100 x oil immersion
objective. I have stained my cells with Nile Red or Draq 7 and I am using the crop function
to get  suitable images of single cells.

I am planning to publish my work, but I have no idea how to obtain a DIC image or suitable
image that will allow me overlay my flourescent image of single cells. Is it possible to use
the crop/zoom function of the software to obtain a DIC image of the same cell at the same
magnification?

Thank you in advance

[George McNamara]

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Hi Shane,

Most LSM510's have a transmitted light detector (PMT-T?). Just turn it
on, adjust the gain and offset. If your LSM has DIC optics, you can put
those in the light path (more on this below), but brightfield is fine
for most scientific needs.

Zoom to the optimum pixel size for resolution needed for your study --
that also enables imaging the fluorophores without photobleaching.
Generally, you just need a single image plane, so bleaching is not a
problem. For confocal 1.4 NA objective lens imaging, I like 60 nm pixel
size (see previous discussions on the confocal listserv). You can use
IRM with very low laser light to find consistent focus (see later).



DIC on Zeiss LSM510, also the Zeiss LSM710 I managed in Miami: Zeiss did
not "nail the landing" on IRM on these confocal microscopes. To get DIC
by eye, the polarizers needed to be in (excitation side) and at 90
degrees on the emission side. This enabled beautiful images (if the
specimen was nice). The LSM510 had a manual polarizer slider that needed
to be slid out manually - which was ok. Where Zeiss "fell flat" is that
the polarizer between the condenser and the PMT-T needed to be manually
moved 90 degrees by the user because the lasers were all linearly
polarizer differntly than the widefield path. All Zeiss had to do was
have the polarizer in the slider be "dropped in" so the two light paths
matched. These are half million dollar (more or less) confocal
microscopes, the crucial light path is the confocal optical path:
getting that perfect is what matters. Even if the by-eye-DIC is not
perfect, doesn't matter.



Consistent focusing tip: IRM is a great way to find the focus of the
coverglass-cell/media interface. If you do not need that focal plane,
you can drive ther microscope Z-focus upwards (I'm assuming inverted
scope) consistently to wherever you need to be. This is much more
consistent than having a biochemist, molecular biologist, cell
biologist, or even microscopist, arbitrarily guessing "best focus" for
each field of view. Or "drive" downwards several um, then up be a few um
(i.e. -8 um then +5 um), to consistently start Z-series at a consistent
plane (-3 um in this example).

Interference reflection microscopy contrast (IRM aka RICM) worked well
on the LSM510 I managed in Miami. Figure 2 of Jiang ... Lossos et al
2010 Blood, panels C and F have confocal IRM images,
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012539/figure/F2/
Dr. Xiaoyu Jiang and I acquired these images on our LSM510 with "0", +2,
and +4 um image planes.


http://www.ncbi.nlm.nih.gov/pubmed/20844236

Jiang X(1), Lu X, McNamara G, Liu X, Cubedo E, Sarosiek KA, Sánchez-García I, Helfman DM, Lossos IS 2010 HGAL, a germinal center specific protein, decreases lymphoma cell motility by
modulation of the RhoA signaling pathway. Blood 116: 5217-5227. doi: 10.1182/blood-2010-04-281568.
Sylvester Comprehensive Cancer Center, University of Miami, Miami, FL, USA.

HGAL is a germinal center (GC)-specific gene that negatively regulates lymphocyte motility and whose expression predicts improved survival of patients with diffuse
large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). We demonstrate that HGAL serves as a regulator of the RhoA signaling pathway. HGAL
enhances activation of RhoA and its down-stream effectors by a novel mechanism - direct binding to the catalytic DH-domain of the RhoA-specific guanine nucleotide
exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate of RhoA. We delineate the structural domain of HGAL that mediates
its interaction with the PDZ-RhoGEF protein. These observations reveal a novel molecular mechanism underlying the inhibitory effects of GC-specific HGAL protein
on the motility of GC-derived lymphoma cells. This mechanism may underlie the limited dissemination and better outcome of patients with HGAL-expressing DLBCL
and cHL.
PMCID: PMC3012539
PMID: 20844236

FYI - the UMiami LSM510 has been (thankfully) retired after many years
of use. An inverted Leica SP5 confocal microscope was moved in. The SP5
IRM has nasty fringes in the middle, but nice quality in the corners -
typical failure of the microscope companies to 'nail the landing'.

IRM images are easy to acquire on widefield microscopes with a filter
cube that does not have an exciter filter (classic Fura-2 dual exciter
cubes for example). On my SOLA-I (which Tom DiMatteo of EpiTechnology,
maker of the CaliCube, http://www.epitechnology.com/contact/, told me at
ABRF 2014 can be make to operate in single-LED mode -- this works with
MetaMorph 7.8.6+ for my first gen SOLA ... feature disabled in later
SOLA-I's) and a "CYR" filter set from Semrock
http://www.semrock.com/SetDetails.aspx?id=2717
CFP/YFP/HcRed-3X-A-000
Filter Role Filter Size Part Number
Single Band Exciter 25 mm x 5.0 mm FF01-427/10-25
<http://www.semrock.com/FilterDetails.aspx?id=FF01-427/10-25>
Single Band Exciter 25 mm x 5.0 mm FF01-504/12-25
<http://www.semrock.com/FilterDetails.aspx?id=FF01-504/12-25>
Single Band Exciter 25 mm x 5.0 mm FF01-589/15-25
<http://www.semrock.com/FilterDetails.aspx?id=FF01-589/15-25>
Triple Band Emitter 25 mm x 3.5 mm FF01-464/542/639-25
<http://www.semrock.com/FilterDetails.aspx?id=FF01-464/542/639-25>
Triple Band Dichroic 25.2 mm x 35.6 mm x 1.1 mm
FF444/521/608-Di01-25x36
<http://www.semrock.com/FilterDetails.aspx?id=FF444/521/608-Di01-25x36>


The IRM is not as nice on my widefield scope as on confocal since
widefield is getting signal from light bouncing off cells etc.

IRM was one of the first super-resolution techniques - the black fringes
show focal adhesions are under 10 nm from the coverglass (probably zero
but there is a longtime debate in the literature and "less than 10" is
still 20x better than fluorescence), and the various grays to white can
be converted to distances by various quantitative IRM methods, such as
dual wavelength qIRM -- which would be even better with 3 wavelengths,
trivial to do on a modern laser scanning confocal microscope. Some IRM
references:

Formation of cell-to-substrate contacts during fibroblast motility: an
interference-reflexion study. <http://www.ncbi.nlm.nih.gov/pubmed/7400245>

*Izzard* CS, *Lochner* LR.

J Cell Sci. 1980 Apr;42:81-116.

PMID:
    7400245


Cell-to-substrate contacts in living fibroblasts: an interference
reflexion study with an evaluation of the technique.
<http://www.ncbi.nlm.nih.gov/pubmed/932106>

*Izzard* CS, *Lochner* LR.

J Cell Sci. 1976 Jun;21(1):129-59.

PMID:
    932106


*Quantitative* reflection interference contrast microscopy (RICM) in
soft matter and cell adhesion. <http://www.ncbi.nlm.nih.gov/pubmed/19816893>

Limozin L, Sengupta K.

Chemphyschem. 2009 Nov 9;10(16):2752-68. doi: 10.1002/cphc.200900601.
Review.

PMID:
    19816893


Absolute interfacial distance measurements by dual-wavelength reflection
interference contrast microscopy.
<http://www.ncbi.nlm.nih.gov/pubmed/14995485>

Schilling J, Sengupta K, Goennenwein S, Bausch AR, Sackmann E.

Phys Rev E Stat Nonlin Soft Matter Phys. 2004 Feb;69(2 Pt 1):021901.
Epub 2004 Feb 12.

PMID:
    14995485


Classic review:

Interference *reflection* microscopy in cell biology: methodology and
applications. <http://www.ncbi.nlm.nih.gov/pubmed/3900106>

*Verschueren H*.

J Cell Sci. 1985 Apr;75:279-301. Review.

PMID:
    3900106


Interference *reflection* microscopy.
<http://www.ncbi.nlm.nih.gov/pubmed/20013754>

Barr VA, Bunnell SC.

Curr Protoc Cell Biol. 2009 Dec;Chapter 4:Unit 4.23. doi:
10.1002/0471143030.cb0423s45.

PMID:
    20013754
    [PubMed - indexed for MEDLINE]

Free PMC Article <http://www.ncbi.nlm.nih.gov/pubmed/20013754>

Many-particle tracking with nanometer resolution in three dimensions by
*reflection interference contrast microscopy*.
<http://www.ncbi.nlm.nih.gov/pubmed/15982050>

Clack NG, Groves JT.

Langmuir. 2005 Jul 5;21(14):6430-5.

PMID:
    15982050

We have developed and characterized a method, based on *reflection
interference contrast microscopy*, to simultaneously determine the
three-dimensional positions of multiple particles in a colloidal
monolayer. To evaluate this method, the interaction of 6.8 microm
(+/-5%) diameter lipid-derivatized silica microspheres with an
underlying planar borosilicate substrate is studied. Measured colloidal
height distributions are consistent with expectations for an
electrostatically levitated colloidal monolayer. The precision of the
method is analyzed using experimental techniques in addition to
computational bootstrapping algorithms. In its present implementation,
this technique achieves 16 nm lateral and 1 nm vertical precision.

Reflection interference contrast microscopy.
<http://www.ncbi.nlm.nih.gov/pubmed/12624905>

Weber I.

Methods Enzymol. 2003;361:34-47. No abstract available.

PMID:
    12624905


*Reflection interference contrast microscopy* of microfilaments in
cultured cells. <http://www.ncbi.nlm.nih.gov/pubmed/6184174>

Opas M, Kalnins VI.

Cell Biol Int Rep. 1982 Nov;6(11):1041-6. No abstract available.

PMID:
    6184174


Adhesions of fibroblasts to substratum during contact inhibition
observed by interference reflection microscopy.
<http://www.ncbi.nlm.nih.gov/pubmed/1169157>

Abercrombie M, Dunn GA.

Exp Cell Res. 1975 Apr;92(1):57-62. No abstract available.

PMID:
    1169157


the beginning:

THE MECHANISM OF ADHESION OF CELLS TO GLASS. A STUDY BY INTERFERENCE
REFLECTION MICROSCOPY. <http://www.ncbi.nlm.nih.gov/pubmed/14126869>

CURTIS AS.

J Cell Biol. 1964 Feb;20:199-215.

PMID:
    14126869
    [PubMed - indexed for MEDLINE]

Free PMC Article <http://www.ncbi.nlm.nih.gov/pubmed/14126869>

The discoverer/inventor of IRM, Prof. A.S.G. Curtis, is still around and
publishing in this field:

Epigenesis: roles of nanotopography, nanoforces and nanovibration.
<http://www.ncbi.nlm.nih.gov/pubmed/24801757>

*Curtis AS*, Tsimbouri PM.

Expert Rev Med Devices. 2014 Jul;11(4):417-23. doi:
10.1586/17434440.2014.916205. Epub 2014 May 7.

PMID:
    24801757

http://informahealthcare.com/doi/abs/10.1586/17434440.2014.916205

//

Also with respect to the super-resolution  2014 Nobel Prize in Chemistry
- researchers in the single particle tracking (SPT) field was tracking
single molecules. 551 PubMed 'hits' for "single particle tracking" (with
quotes), and more by knowing many of the authors. Many were/are
Image-1/AT and MetaMorph customers and/or otherwise connected with
Shinya Inoue and/or Colin Izzard through MBL, Woods Hole.


*Single-particle tracking*: applications to membrane dynamics.
<http://www.ncbi.nlm.nih.gov/pubmed/9241424>

Saxton MJ, Jacobson K.

Annu Rev Biophys Biomol Struct. 1997;26:373-99. Review.

PMID:
    9241424


Protein lateral mobility as a reflection of membrane microstructure.
<http://www.ncbi.nlm.nih.gov/pubmed/8240310>

Zhang F, Lee GM, Jacobson K.

Bioessays. 1993 Sep;15(9):579-88. Review.

PMID:
    824031



Cell migration does not produce membrane flow.
<http://www.ncbi.nlm.nih.gov/pubmed/2211827>

Kucik DF, Elson EL, Sheetz MP.

J Cell Biol. 1990 Oct;111(4):1617-22.

PMID:
    2211827


not all SPT papers use the exact term "single particle tracking"

Nanometer-scale measurements using video light microscopy.
<http://www.ncbi.nlm.nih.gov/pubmed/3141071>

*Schnapp* BJ, *Gelles* J, Sheetz MP.

Cell Motil Cytoskeleton. 1988;10(1-2):47-53.

PMID:
    3141071


Tracking kinesin-driven movements with nanometre-scale precision.
<http://www.ncbi.nlm.nih.gov/pubmed/3123999>

*Gelles* J, *Schnapp* BJ, Sheetz MP.

Nature. 1988 Feb 4;331(6155):450-3.

PMID:
    3123999
... "

The method is applied to measure kinesin-driven bead movements in vitro
with a precision of 1-2 nm.

" ...



Enjoy,


George



On 10/25/2014 10:24 AM, Shane van Breda wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear group members,
>
> I have a very simple question that I require help with. Unfortunately our confocal expert has
> been booked off for three months and I am struggling.
>
> I am using a Zeiss LSM 510 Meta. I am viewing bacteria using a 100 x oil immersion
> objective. I have stained my cells with Nile Red or Draq 7 and I am using the crop function
> to get  suitable images of single cells.
>
> I am planning to publish my work, but I have no idea how to obtain a DIC image or suitable
> image that will allow me overlay my flourescent image of single cells. Is it possible to use
> the crop/zoom function of the software to obtain a DIC image of the same cell at the same
> magnification?
>
> Thank you in advance
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/42

[jerie]

*****
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*****

Shane,

for a true DIC you needed to bring polarizers and prisms into the correct
position, if you just require a transmitted light image of your sample,
activate (check box) the trans detector which will then function like a all
the other detectors,  except there is no pinhole in front of it.

The transaction channel will b fee c cropped like the fluorescent channels.

Hope that helps,  j

http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
Skype jens.Rietdorf
Am 25.10.2014 13:26 schrieb "Shane van Breda" <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear group members,
>
> I have a very simple question that I require help with. Unfortunately our
> confocal expert has
> been booked off for three months and I am struggling.
>
> I am using a Zeiss LSM 510 Meta. I am viewing bacteria using a 100 x oil
> immersion
> objective. I have stained my cells with Nile Red or Draq 7 and I am using
> the crop function
> to get  suitable images of single cells.
>
> I am planning to publish my work, but I have no idea how to obtain a DIC
> image or suitable
> image that will allow me overlay my flourescent image of single cells. Is
> it possible to use
> the crop/zoom function of the software to obtain a DIC image of the same
> cell at the same
> magnification?
>
> Thank you in advance
>

[Shane van Breda]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Thanks for help,

I really appreciate it!

Regards,

Shane

> On 25 Oct 2014, at 8:26 PM, jens rietdorf <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Shane,
>
> for a true DIC you needed to bring polarizers and prisms into the correct
> position, if you just require a transmitted light image of your sample,
> activate (check box) the trans detector which will then function like a all
> the other detectors,  except there is no pinhole in front of it.
>
> The transaction channel will b fee c cropped like the fluorescent channels.
>
> Hope that helps,  j
>
> http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/
> Skype jens.Rietdorf
> Am 25.10.2014 13:26 schrieb "Shane van Breda" <[hidden email]>:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Dear group members,
>>
>> I have a very simple question that I require help with. Unfortunately our
>> confocal expert has
>> been booked off for three months and I am struggling.
>>
>> I am using a Zeiss LSM 510 Meta. I am viewing bacteria using a 100 x oil
>> immersion
>> objective. I have stained my cells with Nile Red or Draq 7 and I am using
>> the crop function
>> to get  suitable images of single cells.
>>
>> I am planning to publish my work, but I have no idea how to obtain a DIC
>> image or suitable
>> image that will allow me overlay my flourescent image of single cells. Is
>> it possible to use
>> the crop/zoom function of the software to obtain a DIC image of the same
>> cell at the same
>> magnification?
>>
>> Thank you in advance
>>

[Ian Dobbie]

*****
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*****

jens rietdorf <[hidden email]> writes:

> Shane,
>
> for a true DIC you needed to bring polarizers and prisms into the correct
> position, if you just require a transmitted light image of your sample,
> activate (check box) the trans detector which will then function like a all
> the other detectors,  except there is no pinhole in front of it.

Do note however that the under objective prism will smear your image in
the splitting direction. It is a small amount 1/3 to 1/2 of your
resolution but in a well setup confocal easily observable. If this
doesn't matter than take all images together. If it does then you need
to do fluorescence first then insert the prism, trying hard not to bash
anything and image in DIC.

Alternatively as George said above, just use brightfield which is
generally sufficient if you just want to know where
cells/nuclei/etc... are.

Ian