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David E. Lewis Department of Chemistry University of Wisconsin-Eau Claire University of Missouri-St. Louis, October 3, 2

Fiat Lux! One Organic Chemist’s Role in Fluorescence Microscopy. David E. Lewis Department of Chemistry University of Wisconsin-Eau Claire University of Missouri-St. Louis, October 3, 2005. Fluorescence. Singlet-singlet transitions Singlet-triplet transition is phosphorescence

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David E. Lewis Department of Chemistry University of Wisconsin-Eau Claire University of Missouri-St. Louis, October 3, 2

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  1. Fiat Lux!One Organic Chemist’s Role in Fluorescence Microscopy David E. Lewis Department of Chemistry University of Wisconsin-Eau Claire University of Missouri-St. Louis, October 3, 2005

  2. Fluorescence • Singlet-singlet transitions • Singlet-triplet transition is phosphorescence • Lifetimes typically less than 1 s • Difference between ex and em is known as Stokes shift • Large Stokes shift is desirable to minimize interference from • Scattering • Indigenous fluorescence model of fluorescence in a diatomic molecule

  3. Preferred probe properties • high selectivity for the target molecule or organelle. • resistant enough to photochemical degradation under normal illumination conditions to permit the target cell feature to be visualized conveniently. • preferably sufficiently non-toxic to allow live cells to be used for the experiment. • highly fluorescent (i.e. it should have a high quantum yield for fluorescence), so that only small amounts of the dye are needed to visualize the cell target of interest. • large Stokes shift to minimize problems from light scattering by the cell • preferably easy to make from readily available, inexpensive starting materials, and chemically stable to permit long-term storage.

  4. The 4-amino-1,8-naphthalimide fluorophore • Photochemically robust • High quantum yields • Chemically easy to manipulate • Low toxicity • Easily delivered to live cells

  5. Fluorescence spectra of a representative 4-amino-1,8-naphthalimide • Large Stokes shifts (≥100 nm) • Why? • Large quantum yield of fluorescence

  6. The eucaryotic cell

  7. Mitochondria are membrane-enclosed organelles distributed through the cytosol of most eukaryotic cells. Their main function is the conversion of the potential energy of food molecules into ATP. Mitochondria have: an outer membrane that encloses the entire structure an inner membrane that encloses a fluid-filled matrix between the two is the intermembrane space the inner membrane is elaborately folded with shelflike cristae projecting into the matrix. a small number (some 5-10) circular molecules of DNA Mitochondria

  8. Key features of the mitochondrion to use in designing a mitochondrial stain • The inner mitochondrial membrane is characterized by • substantial amounts of phosphatidyl serine in the lipid mixture • the presence of a net negative charge on the matrix side of the membrane.

  9. What structural features are needed in the dye? • Delocalized cationic dyes • Sufficient lipohilicity to be membrane-permeant • Cyanines • Mitotracker Green • Triphenylmethane (rhodamine) dyes • reduced dyes • Mitotracker Orange

  10. MitoTracker-type cyanines: four major resonance contributors with complete octets on all atoms; length of delocalized cation system is ≈6-7Å MitoTracker rhodamine-type dyes: four major resonance contributors with complete octets on all atoms; length of delocalized cation system is ≈9.5Å

  11. A potential new mitochondrial probe n = 6 InstantMito LMT-1 n = 4 InstantMito LMT-2 Kristy McNitt

  12. A potential new mitochondrial probe n = 6 InstantMito LMT-1 n = 4 InstantMito LMT-2 But…

  13. Is a 4-dimethylaminopyridinium ion delocalized enough? • Only 2 resonance contributors with complete octets • Length of conjugated, delocalized cation system is only 4.2Å • Most specialists active in fluorescence imaging of cells suggest that this is too short a conjugated system -- too localized -- to successfully cross the intervening membranes

  14. “Actually, yes!” THP-1 monocytes human foreskin fibroblasts Lori Scardino

  15. Confirming that we are localizing in mitochondria MitoTracker® Red: Commercially available mitochondrion dye Colocalization: Yellow areas show where both dyes occupy the same place in the cell InstantMito LMT-1 in THP-1 monocytes

  16. Acidic organelles: Golgi apparatus and lysosomes • Golgi is part of the protein transport system • trans Golgi is moderately acidic (pH ≈ 6.0) • Retrograde transport to Golgi by endocytosis is not uncommon

  17. Lysosomes: the most acidic organelles • Lysosomes are roughly spherical bodies bounded by a single membrane. They are manufactured by the Golgi apparatus (pathway 2 in the figure). They contain over 3 dozen different kinds of hydrolytic enzymes including • proteases • lipases • nucleases • polysaccharidases • The pH within the lysosome is about pH 5, substantially less than that of the cytosol (~pH 7.2). All the enzymes in the lysosome work best at an acid pH. This reduces the risk of their digesting their own cell if they should escape from the lysosome.

  18. What structural features are needed in a lysosome probe? • Dyes that have been used for visualizing lysosomes are almost always • weak bases • membrane-permeant in their unprotonated form • tertiary aliphatic amines • Lysotracker Red

  19. A new lysosomal stain Kristy McNitt Chang, S.-C.; Utecht, R.E.; Lewis, D.E. Dyes Pigments1999, 43, 83-94. InstantLyso LLT-1

  20. How well does it work?

  21. Not bad at all! InstantLyso LLT-1 Color epifluorescence image with live THP-1 monocytes at 75 nM and excited with blue light. Colocalization of InstantLyso LLT-1 and Lysotracker Red in live THP-1 cells; yellow represents colocalized probe. 3D reconstruction of a confocal image series using InstantLyso LLT-1

  22. The accidental discovery: A stain for Golgi apparatus

  23. Recalling the Golgi apparatus • The Golgi apparatus consists of a stack of membrane-bounded cisternae located between the endoplasmic reticulum and the cell surface. A myriad of enzymes (proteins) are present in the Golgi apparatus to perform its various synthetic activities. So there must be mechanisms • to sort out the processed proteins and send them on to their destinations while • reclaiming processing proteins (e.g., glycosylases) for reuse. • pH varies from ≈6.7 in the cis Golgi to ≈6.0 in the trans Golgi

  24. What we were trying to do…

  25. But… • The tosyl chloride was not recrystallized immediately prior to use • The same product was not obtained when freshly recrystallized tosyl chloride was used • We have completely characterized this product as the desired sulfonamide So…

  26. What happened? • Characterization: • The product showed p-toluenesulfonyl resonances in the 1H NMR spectrum • However, the product was not particularly soluble in non-polar solvents • The product did appear to sequester in lysosomes in THP-1 monocytes • Answer:

  27. InstantLyso LLT-1 in fibroblasts BODIPY TR C5 ceramide complexed to BSA Colocalization: Yellow areas show where both dyes occupy the same place in the cell InstantLyso LLT-1 Live foreskin fibroblasts

  28. Targeting cholesterol • Plasma membranes are heterogeneous • Membrane partitions into cholesterol-rich and cholesterol-deficient microdomains • The visualization of cholesterol-rich microdomains of plasma membranes (“rafts”) is carried out in a number of ways. • dehydroergosterol • the pentaene antibiotic, filipin • use of labeled cholera toxin subunit B

  29. A new stain for cholesterol-rich microdomains InstantLipo Sep-1 Kristy McNitt We have also prepared C6 to C18 analogues. These have not all been tested yet, but we know that a minimum of a C8 side chain is required. Chang, S.-C.; Utecht, R.E.; Lewis, D.E. Dyes Pigments1999, 43, 83-94.

  30. It works in live THP-1 monocytes

  31. Confirming that we are localizing in high-cholesterol domains Vybrant® Alexa Fluor® 594: Current state of the art dye for high cholesterol domains Colocalization: Yellow areas show where both dyes occupy the same place in the cell Instant-Lipo Sep-1 Live THP-1 monocytes

  32. And it works in live foreskin fibroblasts… BODIPY TR C5 ceramide complexed to BSA Colocalization: Yellow areas show where both dyes occupy the same place in the cell Instant-Lipo Sep-1

  33. Developing a putative model for the localization cholesterol InstantLipo Sep-1

  34. The similarity of shape of these two molecules cholesterol Two views of the overlay of cholesterol and InstantLipo Sep-1 InstantLipo Sep-1

  35. Gives us a 1:1 model for localization… cholesterol InstantLipo Sep-1 A 1:1 complex of cholesterol and InstantLipo Sep-1

  36. … and another model with more cholesterols We are now attempting a co-crystallization of cholesterol and InstantLipo Sep-1

  37. Other useful properties of some naphthalimide dyes

  38. Medium-dependent fluorescence emission… The loss of the short-wavelength emission as the dielectric constant of the medium increases is demonstrated by InstantLipo Sep-1 in THF-water mixtures. Damon Campbell and Vinay Rao

  39. …which allows us to monitor local water content The dye probably orients itself perpendicular to the membrane surface, with the amino group of the 4-naphthalimide oriented towards the aqueous external phase. The observed fluorescence of InstantLipo Sep-1 in THP-1 monocytes suggests that the local dielectric around the fluorophore is approximately 11.5

  40. Another very medium-sensitive system: fluorescent Tröger’s bases R = n-Bu 57% R = n-C6H13 74% R = n-C8H18 66% Deprez, N.R.; McNitt, K.A.; Petersen, M.E.; Brown, R.G.; Lewis, D.E. Tetrahedron Lett.2005, 46, 2149-2153.

  41. Solvent dependence of Tröger’s base fluorescence

  42. But… It doesn’t cross the cell membrane

  43. Recall -- we want a bleach-resistant stain for microscopy Photochemical bleaching studies

  44. Lysotracker Red -- the benchmark Lysotracker Red at 75 nM in THP-1 cells. Exposures were taken every 5 seconds (with consistent CCD exposure length) with green excitation cube. Unretouched, unprocessed images. Color is already faded extensively by 7 seconds and is nearly gone by 21 seconds. 7 seconds 21 seconds 35 seconds

  45. InstantLyso LLT-1 InstantLyso LLT-1 at 75 nM in THP-1 cells. Exposures were taken every 30 seconds (with consistent CCD exposure length 7.5 seconds) with blue excitation cube (490 nm maximum). Each exposure is some increment of 37.5 seconds. We have skipped the middle group of images. Unretouched, unprocessed images. 0 seconds 75 seconds 338 seconds Betsy Ott and Lori Scardino

  46. The comparison… 0 seconds 75 seconds 7 seconds 21 seconds 35 seconds 338 seconds Lysotracker Red InstantLyso LLT-1

  47. InstantLipo Sep-1 InstantLipo Sep-1 at 200 nM in THP-1 cells. Exposures were taken with consistent CCD exposure length with purple excitation cube. Unretouched, unprocessed images. 5 seconds 35 seconds 65 seconds (For comparison purposes, filipin has faded completely within 15 seconds)

  48. So where to now?

  49. Dyes derivatized with carbohydrates or other interesting biomolecules Robyn Laskowski galactose glucose

  50. Has already led to some unexpected chemistry

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